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Identification of novel avirulence effectors in 

the Dothideomycete plant pathogens, 

Venturia inaequalis and Cladosporium fulvum  

 

 

A thesis presented in partial fulfilment of the 

requirements for the degree of 

 

Doctor of Philosophy (PhD) 

in 

Plant Sciences 

 

 

at Massey University, Manawatū 

New Zealand 

 

 

Silvia de la Rosa  

2022 

 



ii 
 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 



 

iii 
 

Abstract 

Venturia inaequalis and Cladosporium fulvum are important fungal pathogens of 

crop species, causing scab and leaf mould disease of apple and tomato, respectively. 

Resistance to these pathogens is governed by Rvi (apple) and Cf (tomato) resistance (R) 

genes. These R genes encode immune receptors that recognize specific pathogen 

virulence factors, termed avirulence (Avr) effectors, to activate plant defenses. Notably, 

isolates or strains of V. inaequalis and C. fulvum have emerged that can overcome 

resistance mediated by specific R genes in their respective hosts. To better understand 

how these pathogens cause disease or overcome resistance, and to monitor the 

occurrence of resistance-breaking isolates or strains in the field, Avr effectors from 

V. inaequalis and C. fulvum must be identified and functionally characterized. Using a 

combined comparative genomics and phenotyping approach based on progeny from a 

sexual cross between V. inaequalis isolates that differ in their ability to overcome Rvi4 

resistance in apple, a strong candidate for the corresponding AvrRvi4 effector gene was 

identified (Chapter 2). Similarly, using a comparative genomics approach based on in 

planta-expressed effector candidates from C. fulvum strains that differ in their ability to 

overcome Cf-9B resistance in tomato, combined with functional assays, the 

corresponding Avr9B effector gene was identified (Chapter 4). In the resistance-breaking 

isolates or strains studied, the candidate AvrRvi4 gene was disrupted, while the Avr9B 

gene had been deleted. Consistent with most fungal Avr effectors and their genes, both 

the AvrRvi4 candidate and Avr9B are highly expressed in planta, and encode small, 

secreted cysteine-rich proteins. The AvrRvi4 candidate forms part of an expanded 

protein family in V. inaequalis, with members predicted to adopt a β-sandwich fold 

similar to structurally characterized fungal effectors. Avr9B, however, is predicted to 

adopt a novel protein fold. Finally, using a heterologous expression approach, three in 

planta-expressed candidate effectors from V. inaequalis were found to trigger defense 

responses in non-host plants (Nicotiana spp.), suggesting they are recognized by R 

proteins in these species (Chapter 3). Taken together, this thesis has increased our 

understanding of the molecular mechanisms responsible for the activation and 

circumvention of resistance by V. inaequalis and C. fulvum, which will in turn direct host 

cultivar deployment and disease control strategies in the field. 

 



 

iv 
 

 



 
 

v 
 

Acknowledgements 

First and foremost, I would like to thank my supervisor Dr Carl Mesarich, for giving 

me the opportunity to be part of his research team as his first PhD student. I truly value the 

time, effort, and patience you invested in me and my project. I could not forget thanking 

you for all the jokes that brought me into happy tears during lunch time, those moments 

eased the stress I was going through. I also thank my Co-supervisor Prof. Rosie Bradshaw, 

for all her input, suggestions, and guidance. Special thanks to Dr Joanna Bowen for allowing 

me to perform some of my experiments at Plant and Food Research, for offering a place to 

stay in Auckland, for her advice and critical review of my PhD thesis. Thanks also to Saadiah 

Arshed and Brogan McGreal for their contribution to my project regarding bioinformatic 

methods. My gratitude to Dr Vincent Bus for his input and for providing apple seeds, grafted 

trees, and V. inaequalis parent isolates and progeny for my experiments. Thanks to David 

Jones for kindly providing Cf-9 and Cf-9B constructs for my agroinfiltration experiments.  

Thanks to Assoc. Prof. Matthieu Joosten for his valuable feedback and ideas for the 

C. fulvum project, and to Christiaan Schol for his time and effort by completing some of my 

experiments at Wageningen University. My gratitude as well to the collaborators from 

INRAE in France: Bruno LeCam for allowing me to use data from his V. inaequalis isolate EU-

B04 for my screening experiments, and to Mélanie Sannier for sequencing V. inaequalis race 

4 isolates. Thanks to Kim Plummer for her brilliant ideas and her hospitality during my visit 

to the Stromlo Plant Pathology conference in Australia. I would also like to thank the team 

at the MMIC, Matthew Savoian and Raoul Salomon for their help and guidance. 

To the following people that have been part of this long and challenging path, full of 

ups and downs. Without all of you, it would have been almost impossible to have the 

strength and willingness to continue. I am extremely grateful to have shared the lab with 

exceptional scientists, thanks for your friendship, help with my experiments and for being 

there for me in difficult moments; Mariana Tarallo, Ellie Bradley, Mercedes Rocafort, 

Melissa Guo, Simren Brar, Hannah McCarthy, Ashleigh Mosen, Berit Hassing and Lukas 

Hunziker. I’m also grateful for the incredible friends that always had comforting words, an 

advice, or a simple funny anecdote that made me smile; Rayén León, Alejandra Alfaro, Mario 

Alayón, Paul Ogbuigwe and Sebastián Rivera. Special thanks to Carlos Santa Cruz, for 

preparing the best Mexican food and for sharing with me all those delicious meals that made 

me feel like home. Thank you, Diana Cabrera, for always being there for me and for cheering 

me up in decisive moments of my life and PhD.  



 
 

vi 
 

My sincere appreciation to my friends in Mexico and Europe, thanks for your 

friendship, love and support despite the distance and time apart, Beatriz Alvarado, Rolando 

Moreno, Thalia Parra, José Angel Monsivais and Gina Samaniego. Thank you, Dr Felipe 

Reyes, for all your support during both of my scholarship applications, I am sure we will 

meet again.  

To my family: Gracias mamá y papá por su amor, por haberme dado el mejor 

ejemplo, la mejor educación, y por apoyarme siempre en todas mis decisiones. Sin ustedes 

no habría logrado lo que hasta ahora. Los amo tanto y me siento muy afortunada de ser su 

hija. Espero estén orgullosos de mi, esto se los dedico principalmente a ustedes. Espero 

verlos pronto y poder compensar todo este tiempo que he estado tan lejos de casa. Gracias 

a mis tías, primas y sobrinos, especialmente a Brenda Saucedo. Perlita, aún nos quedan 

muchas carcajadas y momentos por compartir, estás en mis pensamientos y confío que vas 

a ganar esta lucha. Admiro tu fortaleza! 



 
 
 

vii 
 

Table of contents 

 

Abstract ....................................................................................................................... iii 

Acknowledgements ....................................................................................................... v 

Table of contents ......................................................................................................... vii 

List of figures ................................................................................................................xii 

List of tables ................................................................................................................ xvi 

List of Abbreviations .................................................................................................. xviii 

Chapter 1: General introduction .................................................................................... 1 

1.1 Plant-pathogen interactions ................................................................................... 4 

1.2 Invasion pattern receptors (IPRs) ............................................................................ 8 

1.2.1 Extracellular IPRs ............................................................................................. 8 

1.2.2 Intracellular IPRs ............................................................................................ 10 

1.3 Features and functions of fungal effector proteins .............................................. 14 

1.3.1 Apoplastic effector proteins .......................................................................... 15 

1.3.2 Cytoplasmic effector proteins ....................................................................... 16 

1.3.3 Avirulence effector proteins .......................................................................... 16 

1.4 The New Zealand horticulture industry ................................................................ 20 

1.5 Cladosporium fulvum ............................................................................................ 22 

1.5.1 Infection cycle of C. fulvum ........................................................................... 22 

1.5.2 Leaf mould control methods ......................................................................... 23 

1.5.3 Molecular aspects of the C. fulvum-tomato interaction ............................... 24 

1.5.4 Cladosporium fulvum effectors ...................................................................... 25 

1.5.5 Tomato Cf resistance proteins ....................................................................... 31 

1.5.6 Avoidance of Cf-mediated resistance ............................................................ 33 

1.6 Venturia inaequalis................................................................................................ 37 

1.6.1 Life cycle of V. inaequalis ............................................................................... 37 

1.6.2 Scab control methods .................................................................................... 39 

1.6.3 Molecular aspects of the V. inaequalis-apple interaction ............................. 41 

1.6.4 V. inaequalis effectors ................................................................................... 44 

1.6.5 Apple Rvi resistance proteins ........................................................................ 47 

1.6.6 Avoidance of Rvi-mediated resistance .......................................................... 47 

1.7 Aims and objectives............................................................................................... 49 



 
 
 

viii 
 

Chapter 2: Identification of the AvrRvi4 avirulence effector from Venturia inaequalis ... 53 

2.1 Introduction ........................................................................................................... 53 

2.2 Materials and methods .......................................................................................... 56 

2.2.1 Biological materials ........................................................................................ 56 

2.2.2 Growth conditions .......................................................................................... 57 

2.2.3 Mating of J222 and NZ203.1 .......................................................................... 58 

2.2.4 V. inaequalis infection assays ......................................................................... 58 

2.2.5 DNA manipulation .......................................................................................... 61 

2.2.6 Genome sequencing ....................................................................................... 62 

2.2.7 Bioinformatic methods ................................................................................... 63 

2.2.8 Allelic variation in the candidate AvrRvi4 gene .............................................. 67 

2.2.9 Structural prediction of the candidate AvrRvi4 effector family ..................... 68 

2.3 Results .................................................................................................................... 69 

2.3.1 Whole genome sequencing provides two new V. inaequalis reference 
genomes ........................................................................................................................ 69 

2.3.2 Mating-type assessment of V. inaequalis isolates ......................................... 70 

2.3.3 SSP comparisons between isolates of V. inaequalis reveal 16 AvrRvi4 effector 
candidates ..................................................................................................................... 71 

2.3.4 Phenotyping of V. inaequalis progeny ........................................................... 75 

2.3.5 Four regions of the V. inaequalis NZ203.1 race (1,4) genome contain single 
nucleotide variants (SNVs) that co-segregate with an inability of the fungus to trigger 
an HR on Rvi4 apple ...................................................................................................... 80 

2.3.6 A single candidate AvrRvi4 gene is present in the NZ203.1 race (1,4) isolate 
genome 82 

2.3.7 The candidate AvrRvi4 gene is surrounded by repetitive elements .............. 89 

2.3.8 The candidate AvrRvi4 gene contains a high level of polymorphism among             
V. inaequalis isolates ..................................................................................................... 89 

2.3.9 V. inaequalis is anticipated to circumvent Rvi4-mediated resistance through 
different mechanisms ................................................................................................... 92 

2.3.10 The candidate AvrRvi4 effector forms part of an expanded family in                               
V. inaequalis .................................................................................................................. 94 

2.3.11 The AvrRvi4 candidate contains a tandemly-repeated promoter element ... 96 

2.3.12 Members of the candidate AvrRvi4 effector family are predicted to adopt a β-
sandwich fold with a large intrinsically disordered region ........................................... 98 

2.3.13 Homologs of the candidate AvrRvi4 effector are restricted to Venturia species 
infecting members of the Malinae subtribe of host plants ........................................ 101 

2.4 Discussion ............................................................................................................. 105 



 
 
 

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Chapter 3: Identification of candidate effector proteins from the apple scab pathogen 

Venturia inaequalis that trigger cell death in non-host plant species .......................... 113 

3.1 Introduction ......................................................................................................... 113 

3.2 Materials and methods ....................................................................................... 116 

3.2.1 Biological materials ...................................................................................... 116 

3.2.2 Growth conditions ....................................................................................... 116 

3.2.3 E. coli and A. tumefaciens electrocompetent cell preparation ................... 117 

3.2.4 DNA manipulation ....................................................................................... 118 

3.2.5 Agrobacterium tumefaciens transformation assays (ATTAs) ...................... 120 

3.2.6 Protein extractions ...................................................................................... 121 

3.2.7 SDS polyacrylamide gel electrophoresis (PAGE).......................................... 121 

3.2.8 Western blotting .......................................................................................... 122 

3.2.9 Selection and bioinformatic analysis of candidate effectors (CEs) from 
Venturia inaequalis .................................................................................................... 122 

3.2.10 Structural prediction of CE proteins ............................................................ 123 

3.2.11 Allelic variation of candidate effectors ........................................................ 124 

3.3 Results ................................................................................................................. 125 

3.3.1 Bioinformatic analysis of CE proteins from Venturia inaequalis ................. 125 

3.3.2 Three CE proteins of V. inaequalis trigger chlorosis and/or cell death in 
Nicotiana species ....................................................................................................... 130 

3.3.3 Apoplastic localization is required for CE proteins of V. inaequalis to trigger 
chlorosis and/or cell death in Nicotiana species ....................................................... 134 

3.3.4 CE proteins of V. inaequalis that trigger chlorosis and/or cell death in 
Nicotiana species are cysteine-rich and encoded by genes that are expressed during 
infection of apple leaves ............................................................................................ 136 

3.3.5 CE proteins of V. inaequalis that trigger cell death in Nicotiana species have 
sequence similarity to effector proteins from plant-pathogenic fungi ..................... 138 

3.3.6 CE proteins of V. inaequalis that trigger cell death in Nicotiana species have 
structural similarity to proteins from plant-pathogenic fungi with characterized tertiary 
structures ................................................................................................................... 143 

3.3.7 CEs that trigger cell death in Nicotiana species are variable in sequence 
between isolates of V. inaequalis .............................................................................. 148 

3.3.8 CE proteins of V. inaequalis do not trigger chlorosis and/or cell death in 
Nicotiana species when targeted to the plant cytoplasm ......................................... 149 

3.4 Discussion ............................................................................................................ 150 

Chapter 4: Identification of the Avr9B avirulence effector from Cladosporium fulvum..159 

4.1 Introduction ......................................................................................................... 159 

4.2 Materials and methods ....................................................................................... 163 



 
 
 

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4.2.1 Biological materials ...................................................................................... 163 

4.2.2 Growth conditions ........................................................................................ 165 

4.2.3 Genomic DNA extraction .............................................................................. 167 

4.2.4 Genome sequencing ..................................................................................... 167 

4.2.5 Identification of candidate Avr9B avirulence effectors ............................... 168 

4.2.6 Tomato infection assays ............................................................................... 169 

4.2.7 RT-qPCR analysis of gene expression ........................................................... 170 

4.2.8 A. tumefaciens infiltration of Nicotiana spp. and tomato ........................... 171 

4.2.9 Gene complementation ............................................................................... 172 

4.2.10 Candidate Avr9B localization vector construction ....................................... 175 

4.2.11 Prediction of Avr9B-C2 tertiary structure .................................................... 176 

4.3 Results .................................................................................................................. 177 

4.3.1 Genome sequencing of C. fulvum IPO 2679 ................................................. 177 

4.3.2 Nucleotide and protein sequence alignments reveal two Avr9B effector 
candidates ................................................................................................................... 177 

4.3.3 Candidate Avr9B avirulence effector genes are expressed during infection of 
tomato 183 

4.3.4 Candidate Avr9 effectors have homologs in other fungal pathogens ......... 184 

4.3.5 A. tumefaciens transient expression transformation assays ....................... 191 

4.3.6 Candidate Avr9B restores avirulence on MM-Cf-9 tomato plants ............... 197 

4.3.7 Avr9B is likely localized to the plant plasma membrane ............................. 200 

4.3.8 Avr9B is predicted to adopt a novel tertiary fold ......................................... 203 

4.4 Discussion ............................................................................................................. 205 

Chapter 5: Discussion and future directions ................................................................ 213 

5.1 Introduction ......................................................................................................... 213 

5.2 Chapter 2: Progress towards the identification of a candidate AvrRvi4 effector from 

V. inaequalis .................................................................................................................... 214 

5.2.1 Future research concerning the candidate AvrRvi4 effector from                              
V. inaequalis ................................................................................................................ 216 

5.3 Chapter 3: Identification of candidate effectors from V. inaequalis that trigger 

defence responses in non-host plants ............................................................................ 220 

5.3.1 Future research concerning CEs of V. inaequalis that trigger defence 
responses in non-host plants ...................................................................................... 222 

5.4 Chapter 4: Identification of the Avr9B effector from C. fulvum .......................... 224 

5.4.1 Future research concerning the Avr9B effector from C. fulvum .................. 226 

5.5 Conclusion ............................................................................................................ 228 



 
 
 

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Appendix ................................................................................................................... 230 

References ……………………………………………………………………………………………………………..….289 

 



 
 
 
 

xii 
 

List of figures 

 

Figure 1.1. Crop diseases caused by fungal pathogens belonging to the Dothideomycetes 

class. .................................................................................................................................. 3 

Figure 1.2. The Zigzag Model of the plant immune system .............................................. 6 

Figure 1.3. The Invasion Model of the plant immune system .......................................... 7 

Figure 1.4. Extracellular invasion patter receptor (IPR) activation and subsequent signal 

transduction in plants ..................................................................................................... 10 

Figure 1.5. Intracellular receptors (NLRs) recognize pathogen effector proteins through 

different strategies .......................................................................................................... 12 

Figure 1.6. Coiled-Coil (CC) and Toll-Interleukin 1 (TIR) nucleotide-binding site (NB) and 

leucine-rich repeat (LRR) receptor (NLR) oligomerize to form a resistosome ............... 13 

Figure 1.7.  Tomato leaf mould disease symptoms and development........................... 23 

Figure 1.8. Crystal structure of the Avr4 effector protein from Cladosporium fulvum...27 

Figure 1.9. Crystal structure of the Ecp6 effector protein from Cladosporium fulvum.. 28 

Figure 1.10. Crystal structure of the Ecp11-1 effector from Cladosporium fulvum ....... 30 

Figure 1.11. Compatible and incompatible Cladosporium fulvum–tomato interactions31 

Figure 1.12. Venturia inaequalis life cycle ...................................................................... 38 

Figure 1.13. Symptoms of scab or blackspot disease on apple plants caused by Venturia 

inaequalis ........................................................................................................................ 39 

Figure 1.14. Apple leaf infection by Venturia inaequalis (compatible interaction)........ 42 

Figure 1.15. Scab reaction classes on apple leaves after inoculation with Venturia 

inaequalis ........................................................................................................................ 44 

Figure 1.16. Venturia inaequalis inside a cellophane membrane as an in culture model 

for in planta growth ........................................................................................................ 46 

Figure 2.1. Arrangement of Venturia inaequalis isolates in the phenotyping experiment 

on detached leaves of apple from a cross between cultivar ‘Royal Gala’ and accession 

TSR33T239 carrying the Rvi4 resistance gene ................................................................ 60 

Figure 2.2. Cumulative genome length as a function of contig number and size for the 

parental isolates J222 and NZ203.1 of Venturia inaequalis ........................................... 69 



 
 
 
 

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Figure 2.3. Presence and absence of the alpha box associated with mating-type gene 

MAT1-1  in Venturia inaequalis isolates J222 and NZ203.1 ............................................ 70 

Figure 2.4. Macroscopic observations of Venturia inaequalis isolates inoculated on Rvi4 

apple leaves.. ................................................................................................................... 76 

Figure 2.5. Microscopic observations of Venturia inaequalis isolates inoculated on Rvi4 

apple leaves. .................................................................................................................... 77 

Figure 2.6. Schematic representation of the atg6122 (candidate AvrRvi4) gene from 

Venturia inaequalis across four isolates ......................................................................... 84 

Figure 2.7. Nucleotide sequence of the candidate AvrRvi4 gene from Venturia inaequalis 

and the protein it encodes .............................................................................................. 88 

Figure 2.8. Genomic location of the candidate AvrRvi4 gene ........................................ 89 

Figure 2.9. Sequence variation in the candidate AvrRvi4 gene across a worldwide 

collection of Venturia inaequalis f. sp. pomi, V. inaequalis f. sp. pyracanthae and                  

V. inaequalis f. sp. eriobotryae isolates .......................................................................... 91 

Figure 2.10. Alignment of proteins from the candidate AvrRvi4 effector family of 

Venturia inaequalis MNH120 .......................................................................................... 95 

Figure 2.11. Expression levels of the candidate AvrRvi4 effector gene family from 

Venturia inaequalis  isolate MNH120. ............................................................................ 96 

Figure 2.12. Tandem repeats present in the promoter region of the candidate AvrRvi4 

effector gene from Venturia inaequalis .......................................................................... 97 

Figure 2.13. Predicted tertiary structure of candidate AvrRvi4 effector family members 

from Venturia inaequalis............................................................................................... 100 

Figure 2.14. Surface charge potential of the candidate AvrRvi4 effector protein from 

Venturia inaequalis ....................................................................................................... 101 

Figure 2.15. Alignment of homologs of the candidate AvrRvi4 effector family in Venturia 

species. .......................................................................................................................... 103 

Figure 2.16. Phylogenetic tree of the AvrRvi4 effector family and homologs in other 

Venturia species ............................................................................................................ 104 

Figure 3.1. Clustal Ω protein alignment of Venturia inaequalis (MNH120) candidate 

effector 10 (CE10) with similar proteins from other fungi ........................................... 127 

Figure 3.2.  Clustal Ω protein alignment of Venturia inaequalis (MNH120) candidate 

effector 11 (CE11) with similar proteins from other fungi. .......................................... 128 



 
 
 
 

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Figure 3.3. Clustal Ω protein alignment of Venturia inaequalis (MNH120) candidate 

effectors 12 (CE12) and 13 (CE13) to similar proteins from members of the Venturia 

genus ............................................................................................................................. 129 

Figure 3.4. Three candidate effectors (CEs) of Venturia inaequalis trigger chlorosis 

and/or cell death in the model non-host species Nicotiana benthamiana (Nb) and 

Nicotiana tabacum (Nt) using an Agrobacterium tumefaciens-mediated transient 

expression assay (ATTA) ................................................................................................ 131 

Figure 3.5. Apoplastic localization is required for candidate effector (CE) proteins of 

Venturia inaequalis to trigger chlorosis and/or cell death in Nicotiana benthamiana (Nb) 

and Nicotiana tabacum (Nt) ......................................................................................... 135 

Figure 3.6. Candidate effectors (CEs) of Venturia inaequalis (MNH120) that trigger 

chlorosis and/or cell death in the model non-host species Nicotiana benthamiana and 

Nicotiana tabacum are small secreted cysteine-rich proteins, and are encoded by genes 

that are expressed in planta ......................................................................................... 137 

Figure 3.7. Candidate effector 17 (CE17) of Venturia inaequalis (MNH120) has sequence 

similarity to proteins from other fungi ......................................................................... 140 

Figure 3.8. Candidate effector 93 (CE93) of Venturia inaequalis (MNH120) has sequence 

similarity to hypothetical proteins from other fungi .................................................... 141 

Figure 3.9. Candidate effector 126 (CE126) of Venturia inaequalis (MNH120) has 

sequence similarity to proteins from Dothideomycete fungi....................................... 142 

Figure 3.10. Predicted tertiary structure of Candidate Effector 17 (CE17) from Venturia 

inaequalis. ..................................................................................................................... 145 

Figure 3.11.  Predicted structure of candidate effector 93 (CE93) from Venturia 

inaequalis ...................................................................................................................... 146 

Figure 3.12.  Predicted structure of candidate effector 126 (CE126) from Venturia 

inaequalis ...................................................................................................................... 147 

Figure 3.13. Sequence variation for three candidate effector (CE) genes across isolates 

of Venturia inaequalis ................................................................................................... 148 

Figure 4.1. The 36.5 kb Hcr9 (Homologues of Cladosporium fulvum resistance gene Cf-9) 

introgression segment from wild tomato species Solanum pimpinellifolium .............. 161 

Figure 4.2. Schematic representation of the two candidate Avr9B avirulence effector 

proteins from strain 0WU of Cladosporium fulvum...................................................... 182 



 
 
 
 

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Figure 4.3.  Presence-or-absence of Avr9B-C2 across Cladosporium fulvum strains 

collected from around the world .................................................................................. 183 

Figure 4.4. The Avr9B candidate genes are strongly induced in planta, relative to 

expression in culture ..................................................................................................... 184 

Figure 4.5.  Avr9B-C1 from Cladosporium fulvum has similarity to uncharacterized 

proteins present in other fungal plant pathogens ........................................................ 187 

Figure 4.6. Avr9B-C2 from Cladosporium fulvum has similarity to uncharacterized 

proteins present in other fungal plant pathogens (Group 1) ....................................... 188 

Figure 4.7. Avr9B-C2 from Cladosporium fulvum has similarity to uncharacterized 

proteins present in other fungal plant pathogens (Group 2) ....................................... 190 

Figure 4.8.  Co-expression of candidate Avr9B avirulence effectors Avr9B-C1 or Avr9B-

C2 from Cladosporium fulvum with the Cf-9 or Cf-9B R proteins of tomato in the model 

non-host species Nicotiana tabacum using Agrobacterium tumefaciens-mediated 

transient transformation assays (ATTAs) ...................................................................... 193 

Figure 4.9. Co-expression of Avr9B-C2 homologs from the tomato pathogens 

Stemphylium lycopersici and Pseudocercospora fuligena with the Cf-9 or Cf-9B R proteins 

of tomato in the model non-host species Nicotiana tabacum using Agrobacterium 

tumefaciens-mediated transient transformation assays (ATTAs) ................................ 194 

Figure 4.10. A cysteine to serine substitution at position 77 is indispensable for the 

Avr9B-C2 chlorotic response, as well as to trigger a Cf-9B-dependent hypersensitive 

response. ....................................................................................................................... 196 

Figure 4.11. Strain IPO 2679 of Cladosporium fulvum complemented with Avr9B-C1 or 

Avr9B-C2. ....................................................................................................................... 198 

Figure 4.12. Avr9B-C2 of Cladosporium fulvum strain 0WU restores avirulence on MM-

Cf-9 ................................................................................................................................ 199 

Figure 4.13. In planta localization of mCherry-Avr9B. .................................................. 201 

Figure 4.14. In planta localization of GFP-Avr9B .......................................................... 202 

Figure 4.15. Plasmolysis of Nicotiana tabacum cells expressing GFP-Avr9B ................ 203 

Figure 4.16. Predicted tertiary structure of Avr9B from Cladosporium fulvum ........... 204 



 
 
 

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List of tables 

 

Table 1.1. Identified Avirulence (Avr) effector proteins from plant-pathogenic 

Dothideomycete fungi and their corresponding plant resistance (R) proteins. ............. 18 

Table 1.2. New Zealand apple industry overview ........................................................... 20 

Table 1.3. New Zealand tomato industry overview ........................................................ 21 

Table 1.4. Features of tomato Cf resistance (R) proteins, mediating resistance to 

Cladosporium fulvum. ..................................................................................................... 32 

Table 1.5. Allelic variation in the avirulence (Avr) and extracellular protein (Ecp) effector 

genes of Cladosporium fulvum. ...................................................................................... 34 

Table 1.6. Scab immune receptor (R) genes in apple and their corresponding avirulence 

(Avr) effector genes from Venturia inaequalis. .............................................................. 41 

Table 2.1. Biological materials used in Chapter 2 ........................................................... 56 

Table 2.2. Genome assembly statistics for Venturia inaequalis parental isolates J222 and 

NZ203.1 ........................................................................................................................... 69 

Table 2.3. Mating-type gene present in the parental isolates J222 and NZ203.1 of 

Venturia inaequalis, as well as 50 progeny (S001–S107) from the J222 x NZ203.1 cross..

 ......................................................................................................................................... 71 

Table 2.4. AvrRvi4 avirulence effector candidates identified through a small secreted 

protein (SSP) sequence comparison across isolates of Venturia inaequalis. ................. 74 

Table 2.5. Prediction of which progeny (S001–S107) from a cross between parental 

isolates J222 and NZ203.1 of Venturia inaequalis contain a functional copy of the AvrRvi4 

effector gene ................................................................................................................... 78 

Table 2.6. Summary of regions in the Venturia inaequalis NZ203.1 race (1,4) genome that 

have the best correlation between single nucleotide variants (SNVs) and an inability of 

progeny from a cross between isolates J222 and NZ203.1 to trigger a hypersensitive 

response (HR) on Rvi4 apple. .......................................................................................... 81 

Table 2.7 Correlation between the genotype of Venturia inaequalis J222 race 

(1)/NZ203.1 race (1,4)/J222 x NZ203.1 progeny isolates and their phenotype on Rvi4 

apple. ............................................................................................................................... 85 

Table 2.8. Allelic variation in the candidate AvrRvi4 gene and the protein it encodes 

across isolates of Venturia inaequalis that infect apple, loquat and pyracantha .......... 90 



 
 
 

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Table 2.9. Polymorphisms in candidate AvrRvi4 genes from three Venturia inaequalis 

isolates overcoming Rvi4-mediated resistance. ............................................................. 93 

Table 3.1. Biological materials used in Chapter 3 ......................................................... 116 

Table 3.2. Allelic variation in three candidate effector (CE) genes of Venturia inaequalis

 ....................................................................................................................................... 149 

Table 4.1.  Bacterial and fungal strains, and plant material used in Chapter 4. ........... 163 

Table 4.2 DNA polymorphisms in the coding sequences of small secreted proteins (SSPs) 

from New Zealand Cladosporium fulvum strain IPO 2679 with respect to C. fulvum 

reference strain 0WU. ................................................................................................... 179 

Table A.1 List of primers used in this thesis…………………………………………………………….…230 

Table A.2 List of plasmids used in this thesis……………………………………………………………..233 

Table A.3 Regions in the Venturia inaequalis NZ203.1 race (1,4) genome that have the 

best correlation between single nucleotide variants (SNVs) and an inability of progeny 

from a cross between isolates J222 and NZ203.1 to trigger a hypersensitive response 

(HR) on Rvi4 apple……………………………………………………………………………………………………246 

Table A.4 Venturia inaequalis isolates assessed for allelic variation analyses……………248 

Table A.5 Bioinformatic analysis of 133 candidate effectors (CEs) from Venturia 

inaequalis…………………………………………………………………………………………………………………264 

 

 

 

 

 

 

 

 

 

 

 

 



 
 
 

xviii 
 

List of Abbreviations 

aa amino acid(s) 

ADPR adenosine diphosphate ribose 

AF apoplastic fluid 

Amp ampicillin 

APS ammonium persulfate 

ATP adenosine triphosphate 

ATR1 Arabidopsis thaliana Recognized 1 

ATTA Agrobacterium tumefaciens transformation assay(s) 

Avr avirulence 

BAC bacterial artificial chromosome 

BAK1 Brassinosteroid Insensitive 1-Associated Kinase 1 

bp base pair(s) 

BLAST basic local alignment search tool 

BSA bulk segregant analysis 

°C degrees Celsius 

CC coiled-coil  

CBM carbohydrate-binding module 

cDNA complementary DNA 

cds coding sequence 

CfCEs Cladosporium fulvum candidate effectors 

CE candidate effector 

Cin cellophane-induced 

cm centimetre(s) 

CPPs cerato-platanin(s) 

CTAB cetyltrimethyl ammonium bromide 

CWDEs  cell wall-degrading enzymes  

Cys  cysteine  

DAMP damage-asssociated molecular pattern 

DMI demethylation inhibitor 

DMSO dimethyl sulfoxide 

DNA deoxyribunocleic acid 

dNTP deoxyribonucleic triphosphate 

dpi days post-inoculation 

DTT dithiothreitol 

ECPs extracellular proteins 

ED ectodomain 

EDTA ethylenediaminetetraacetic acid 

ER endoplasmic reticulum 



 
 
 

xix 
 

ETI effector-triggered immunity 

ETS effector-triggered susceptibility   

fgl flagellin 

FLS2 flagellin-sensing 2  

FPMK fragments per million per kilobase 

g gram 

g gravitational force 

GFP green fluorescent protein 

GH glycosyl hydrolase 

GPI glycosylphosphatidylinositol 

GWAS genome-wide association study 

h hour(s) 

ha hectare  

HABS high-affinity binding sites 

HR hypersensitive response 

ID integrated domain 

IDR intrinsically disordered region 

IM induction medium 

IP invasion pattern 

IPR invasion pattern receptor 

IPTG Isopropyl β-D-1-thiogalactopyranoside 

IPTR invasion pattern-triggered response  

JGI Joint Genome Institute 

KD kinase domain 

kDa kilodalton(s) 

L litres 

LB lysogeny broth 

LPMOs lytic polysaccharide monooxygenase(s) 

LPS lipopolysaccharide 

LRR leucine-rich repeat 

LysM lysin motif 

M molar 

MAMP microbe-associated molecular pattern  

MAPK mitogen-activated protein kinase  

MBC methyl benzimidazole carbamate 

mg milligram 

min minute(s) 

ml millilitre(s) 

mm millimetre(s) 

mM millimolar 



 
 
 

xx 
 

MM minimal medium 

MMT million metric tons 

MS Murashige and Skoog  

MTI MAMP-triggered immunity  

MW molecular weight 

NB nucleotide-binding site  

NCBI National Centre for Biotechnology Information 

NEP1 necrosis and ethylene-inducing peptide 1  

NGS next generation sequencing 

NLPs necrosis and ethylene-inducing peptide 1-like proteins  

NLS nuclear localization signal 

nt nucleotide(s) 

NZ New Zealand 

µg microgram(s) 

µl microlitre(s) 

µM micromolar 

OD optical density 

PAGE polyacrylamide gel electrophoresis 

PAMP pathogen-associated molecular pattern 

PCR polymerase chain reaction 

PDA potato dextrose agar 

PDB protein data bank 

PDB potato dextrose broth 

PEP1 protein essential during penetration 1 

pLDDT predicted local-distance difference test 

PNPL plant natriuretic peptide-like 

PR pathogenesis-related 

PR1α pathogenesis-related protein 1α 

PRR pattern recognition receptor 

PTI PAMP-triggered immunity 

PVDF polyvinylidene fluoride 

PVP polyvinylpyrrolidone 

PVX potato virus X 

QoI quinone outside inhibitor 

QTL quantitative trait locus 

R resistance 

RCPs repeat-containing protein(s) 

RH relative humidity 

RIP repeat-induced mutation 



 
 
 

xxi 
 

RiPPs ribosomally and post-translationally modified 

peptide(s) 

RLCKs receptor-like cytoplasmic kinase(s) 

RLKs receptor-like kinase(s) 

RLPs receptor-like protein(s) 

RNA ribonucleic acid 

ROS reactive oxygen species  

rpm revolutions per minute 

RPP1 recognition of Peronospora parasitica 1 

RT room temperature 

RT-qPCR quantitative reverse transcription PCR 

SA salicylic acid 

SDHI succinate dehydrogenase inhibitor 

SDS sodium dodecyl sulfate 

sec second(s) 

SERK3 somatic embryogenesis receptor kinase 3  

SNPs single nucleotide polymorphism(s) 

SNVs single nucleotide variant(s) 

SOBIR1 suppressor of BAK1-interacting receptor-like kinase 1   

SP signal peptide 

SRA sequence read archive 

SSPs small secreted protein(s) 

TAE tris-acetic acid EDTA 

TBST tris-buffered saline and Tween 20  

TBSV tomato bushy stunt virus  

T-DNA transfer DNA 

TEMED tetramethylethylenediamine 

TIR toll/interleukin-receptor 

TM transmembrane domain 

UV ultraviolet  

V volt(s) 

VICE Venturia inaequalis candidate effector 

VIGS virus-induced gene silencing 

WA water agar 

X-gal 5-bromo-4-chloro-3indolyl-β-D-galactopyranoside 

XopQ Xanthomonas outer protein Q 

YFP yellow fluorescent protein 

 





Chapter 1: General introduction 
 
 

1 
 

Chapter 1:  General introduction 
 

Fungal plant pathogens are a global threat to food security as they cause diverse 

crop diseases that result in high agricultural yield losses each year. Estimations of global 

annual agricultural production losses from fungal diseases range from 10 to 23% 

(Steinberg & Gurr, 2020) and in some cases can reach up to 30% (Savary et al., 2019). 

Such losses are anticipated to increase with climate change, as the warmer and wetter 

weather conditions favour disease, but also the spread of fungal species further north 

into the Northern hemisphere and further south into the Southern hemisphere (Bebber 

et al., 2013; Chaloner et al., 2021). Many diseases affecting and threatening crops are 

caused by fungal pathogens belonging to the phylum Ascomycota, and in particular, the 

Dothideomycetes, which is the largest and most ecologically diverse class in the kingdom 

Fungi  (Haridas et al., 2020; Kirk et al., 2008). The Dothideomycete class can be divided 

into more than 23 orders comprised of 110 families, 1,261 genera and about 19,000 

species (Wijayawardene et al., 2017). The Pleosporales, Capnodiales and Venturiales are 

three Dothideomycete orders that include many of the most devastating pathogens 

worldwide (Ohm et al., 2012). Among the main crop diseases caused by these orders of 

fungi are Septoria nodorum blotch of wheat (caused by Parastagonospora nodorum) 

(Quaedvlieg et al., 2013), net blotch of barley (caused by Pyrenophora teres f. maculata) 

(Backes et al., 2021), blackleg disease of brassicas (caused by Leptosphaeria maculans) 

(Becker et al., 2017), scab disease of apple (caused by Venturia inaequalis) (Bowen et 

al., 2011), leaf mould disease of tomato (caused by Cladosporium fulvum) (Mesarich et 

al., 2018),  black sigatoka disease of banana (caused by Pseudocercospora fijiensis) 

(Arango et al., 2016), and septoria leaf blotch of wheat (caused by Zymoseptoria tritici) 

(Kildea et al., 2021) (Fig. 1.1). Depending on the fungus, the lifestyle of these 

Dothideomycetes can be biotrophic, necrotrophic or hemibotrophic. Biotrophs, such as 

V. inaequalis and C. fulvum, depend on living tissue to acquire nutrients and colonize 

their hosts. Hemibiotrophs, such as L. maculans, P. fijiensis and Z. tritici initially have a 

biotrophic phase, where they require living host tissue, but then later shift to a 

necrotrophic lifestyle, whereby they kill the host to obtain nutrients. Finally, 

necrotrophs, such as P. nodorum and P. teres f. maculata, steadily decompose their host 



Chapter 1: General introduction 
 
 

2 
 

cells to obtain nutrients for growth (Pradhan et al., 2021; Vleeshouwers & Oliver, 2014). 

It should be noted, however, that some biotrophic pathogens are sometimes referred 

to as hemibiotrophs, despite not having a necrotrophic phase. Indeed, the term 

hemibiotroph is also often used to describe pathogens that switch from a biotrophic to 

saprophytic mode of growth, such as V. inaequalis (Bowen et al., 2011). C. fulvum, too, 

is also sometimes referred to as a hemibiotroph (e.g., Ökmen et al., 2017). This is 

because, under some conditions, disease lesions become chlorotic or necrotic late in the 

infection cycle. This is thought to be due to the production and aggregation of 

conidiophores that block stomata, limiting respiration, that in severe infections, leads to 

the death of the host (Thomma et al., 2005).   

 

 

 

 

 

 

 

 

 

 

 

 

 

 



Chapter 1: General introduction 
 
 

3 
 

 
Figure 1.1. Crop diseases caused by fungal pathogens belonging to the Dothideomycetes class. (A) Septoria nodorum blotch disease of wheat caused by 

Parastagonospora nodorum (Department of Agriculture and Food Western Australia, DAFWA (2013)). (B) Net blotch disease of barley caused by Pyrenophora teres 

f. maculata (Geoff Thomas, DAFWA (2021)). (C) Blackleg disease of Brassicas caused by Leptosphaeria maculans (Justin Kudnig, Advanta Seeds (2018)). (D) Scab or 

black spot disease of apple caused by Venturia inaequalis (Department of Primary Industries and Regional Development, DPIRD (2021)). (E) Leaf mould disease of 

tomato caused by Cladosporium fulvum (Nexles® EU). (F) Black Sigatoka disease of banana caused by Pseudocercospora fijiensis (Prof. André Drenth, University of 

Queensland). (G) Septoria leaf blotch disease of wheat caused by Zymoseptoria tritici (South Australian Research and Development Institute (1992)).



Chapter 1: General introduction 
 
 

4 
 

1.1 Plant-pathogen interactions 

To cause disease, plant-pathogenic fungi must enter their hosts. To do this, they 

first need to circumvent preformed physical and chemical barriers at the plant surface. 

Physical barriers include stomata, the cuticle and in some instances, the cell wall (Wang 

& Wang, 2018),  whereas chemical barriers include phytoanticipins, which are secondary 

metabolites stored in plant tissues (Zaynab et al., 2018). Such barriers serve as a basic 

level of defence against invading organisms. Thus, as soon as contact is made, 

pathogenic fungi sense such physical and chemical components on the plant surface 

(e.g. stomatal pores, wax, cellulose and cutin), to stimulate the processes of germination 

and penetration required for host entry (Lo Presti et al., 2015; Toruno et al., 2016). The 

Dothideomycetes Alternaria alternata and Alternaria brassicicola, as well as the 

Sordariomycete Fusarium solani, for example, deploy cutinases to penetrate the cuticle, 

whereas L. maculans and C. fulvum use the topography signals of stomatal pores and 

wounds to enter into leaves (Asai & Shirasu, 2015; Kim et al., 1998; Stotz et al., 2014; 

Tanaka et al., 2017). Some fungi use specialized infection structures named appressoria 

to penetrate the plant tissue. To penetrate, the appressorium must adhere firmly to the 

host surface and form an infection peg that breaks into the cuticle and cell wall (Huang, 

2001). V. inaequalis can breach the cuticle using appressoria combined with enzymatic 

activity (Koller et al., 1991). Contrarily, the Basidiomycota fungus Ustilago maydis and 

the Sordariomycete Colletotrichum higginsianum, use appressoria to accumulate turgor 

pressure and facilitate the entry into the host through mechanical force (Giraldo & 

Valent, 2013; Ryder & Talbot, 2015).  

Once physical and pre-formed chemical barriers have been breached or overcome 

by plant-pathogenic fungi, plants must rely on their immune system to provide 

protection against infection and disease. Over recent years, several models have been 

put forward to describe the plant immune system. The most well-known of these is the 

Zigzag Model (Fig. 1.2) (Jones & Dangl, 2006). This model separates the plant immune 

system into two layers; the first layer relies on pattern recognition receptors (PRRs) at 

the cell surface, which recognize conserved pathogen-associated molecular patterns 

(PAMPs), such as chitin in fungi or flagellin of bacteria to give PAMP-triggered immunity 

(PTI). PTI defence responses include callose deposition, production of reactive oxygen 

species (ROS), ion fluxes at the plasma membrane, accumulation of defence hormones 



Chapter 1: General introduction 
 
 

5 
 

and antimicrobial compounds, and the expression of other pathogenesis-related (PR) 

genes (Boller & Felix, 2009; Couto & Zipfel, 2016; Giraldo & Valent, 2013). To successfully 

infect their hosts, invading plant pathogens must then deliver virulence factors, termed 

effectors, into host cells to suppress PTI to give effector-triggered susceptibility (ETS). If 

the pathogen does not have the right toolkit of effector proteins, then PTI cannot be 

overcome. In the second layer of the plant immune system, certain plants have evolved 

to recognize one or more of these effector proteins to trigger effector-triggered 

immunity (ETI), rendering the plant resistant. This recognition relies on intracellular 

immune receptors called resistance (R) proteins, with the recognized effectors termed 

avirulence (Avr) effector proteins, as the pathogen is rendered avirulent. ETI is 

characterized by a strong localized cell death reaction at the pathogen infection site, 

termed the hypersensitive response (HR), which halts the growth of the pathogen 

(Heath, 2000). To successfully cause infection and disease, plant pathogens then need 

to suppress ETI through an expansion of their effector repertoire or through, for 

example, deletion or modification of the gene encoding the recognized Avr effector, 

such that recognition can no longer occur (Jones & Dangl, 2006). These interactions 

between R and Avr proteins are the ongoing result of a molecular arms race between 

plant pathogens and their hosts to evade and regain recognition, respectively. 

Over recent years, however, it has become increasingly clear that the Zigzag 

Model has several key shortfalls. For example, the Zigzag Model does not accommodate 

fungal pathogens with necrotrophic or hemibiotrophic lifestyles. Furthermore, it does 

not acknowledge the involvement of endogenous damage-associated molecular 

patterns (DAMPs) (e.g. plant cell wall fragments released by fungal cell wall-degrading 

enzymes) in triggering PTI, or the fact that some PTI responses are strong, while some 

ETI responses are weak or do not involve an HR. Notably, the distinction between PAMPs 

and effectors has also become increasing ambiguous, with a number of conserved 

effectors from plant-pathogenic fungi now known to be recognized by PRRs as PAMPs 

(Boller & Felix, 2009; Thomma et al., 2011). PRRs, too, have been shown to function as 

R proteins outside of the cell, where they recognize effector proteins from, for example, 

C. fulvum, to trigger ETI (see section 1.5.5). With these points in mind, an alternative 

view of the plant immune system, called the Invasion Model was proposed (Fig. 1.3) 

(Cook et al., 2015). This model states that molecules with any function, be that a PAMP, 



Chapter 1: General introduction 
 
 

6 
 

effector or DAMP, are collectively called invasion patterns (IPs). IPs are perceived by 

invasion pattern receptors (IPRs) which include both extracellular and intracellular 

immune receptors, leading to IP-triggered responses (IPTRs) of various strengths, 

depending on the IP and IPR involved. The outcome of an IPTR can either be the 

continuation or the end of the symbiosis (infection) due to the success or failure to 

suppress IPTR, respectively. 

 

 

Figure 1.2. The Zigzag Model of the plant immune system. In phase 1, the plant immune system 

recognizes and responds to conserved pathogen-associated molecular pattern (PAMPs), via 

pattern recognition receptors (PRRs) leading to PAMP-triggered immunity (PTI). In phase 2, 

pathogens secrete effectors to suppress PTI, or enable pathogen nutrition and dispersal, 

resulting in effector-triggered susceptibility (ETS). In phase 3, an effector is recognized by an 

intracellular resistance (R) protein to trigger effector-triggered immunity (ETI) that results in a 

hypersensitive cell death response (HR). The recognized effector is termed an avirulence (Avr) 

protein. In phase 4, pathogens have lost, mutated, or gained new effectors through horizontal 

gene flow (in blue), to suppress ETI. Selection favors new plant R protein alleles that can 

recognize the altered or new effectors, resulting again in ETI. Figure from Jones & Dangl (2006). 



Chapter 1: General introduction 
 
 

7 
 

 

 

 

Figure 1.3. The Invasion Model of the plant immune system. Invasion patterns (IPs), whether 

pathogen-associated molecular patterns (PAMPs), damage-associated molecular patterns 

(DAMPs) or effectors, are perceived by plant invasion pattern receptors (IPRs), leading to an IP-

triggered defence response (IPTR). In the case of biotrophs, this IPTR may stop the symbiosis 

(infection). Invading pathogens however can use effectors to suppress IPTRs. In the case of 

necrotrophs, these can utilize the IPTR for their own advantage (e.g. nutrient acquisition) and 

continue the symbiosis. Figure from Cook et al. (2015).  

 

 

From this point forward, I will use the term Avr effector in the context of being 

an IP, extracellular and intracellular R proteins in the context of being an IPR, and HR or 

another R protein-mediated response in the context of being an IPTR, consistent with 

the Invasion Model.  

 At the simplest level, the interaction between pathogen Avr effector proteins 

and plant R proteins is governed by pathogen Avr effector genes and plant R genes, and 

is best described by the Gene-for-Gene Model proposed by Flor (1971). This model 

suggests that the occurrence of matching genes in the host (R genes) and pathogen (Avr 

genes) are required for host resistance, leading to incompatibility. Furthermore, this 

model points out that the absence, alteration or loss of either the R or Avr gene 

compromises host resistance, leading to disease or compatibility. However, Avr–R 



Chapter 1: General introduction 
 
 

8 
 

interactions are not always this simple. Complex interactions between Avr and R 

proteins have been described in cases where two Avr proteins are recognized by a single 

R protein, when an Avr protein is recognized by more than one R protein, and when 

another effector is able to mask the presence of another Avr protein (Anh et al., 2018; 

Ghanbarnia et al., 2018; Petit-Houdenot et al., 2019).  

For example, the Avr gene encoding AvrLm10 from L. maculans and the R gene 

encoding Rlm10 from Brassica nigra do not interact in a typical gene-for-gene manner, 

but instead display a 'genes-for-gene' interaction (Petit-Houdenot et al., 2019). More 

specifically, the AvrLm10 avirulence is conferred by two adjacent Avr genes, AvrLm10A 

and AvrLm10B, which are both necessary to trigger Rlm10-mediated resistance (Petit-

Houdenot et al., 2019). In another example, a 'gene-for genes' interaction is observed 

between the Avr gene AVR–Rmg8 from the Sordariomycete Pycularia oryzae and the 

wheat R genes Rmg8 and Rmg7, which encode R proteins that can both recognize the 

Rmg8 Avr effector (Anh et al., 2018). Finally, the L. maculans gene encoding the    

AvrLm4-7 effector protein can block the recognition of the AvrLm5-9 Avr protein by the 

corresponding B. napus Rlm9 resistance protein (Ghanbarnia et al., 2018). To the 

contrary, necrotrophic fungi follow the Inverse Gene-for-Gene Model. Under this model, 

fungi instead have effector genes that encode host-selective toxins (HSTs). These HSTs 

interact with the product of plant susceptibility (S) genes to cause disease susceptibility 

instead of resistance (Friesen et al., 2007). These examples illustrate how complex and 

dynamic the interactions between plants and fungal pathogens are. In both cases, the 

Avr and HST can be thought of as IPs and the R and S proteins as IPRs.  

1.2 Invasion pattern receptors (IPRs) 

Activation of plant immune responses depend on the recognition of IPs by 

extracellular or intracellular IPRs.  

1.2.1 Extracellular IPRs 

Extracellular IPRs are highly conserved transmembrane proteins that are activated 

upon recognition of apoplastic IPs. PRRs are divided into main two classes: the receptor-

like kinases (RLKs) and the receptor-like proteins (RLPs) (Zipfel, 2014). RLKs possess a 

ligand-binding domain (ectodomain), a transmembrane (TM) domain and a cytoplasmic 



Chapter 1: General introduction 
 
 

9 
 

kinase domain, whereas RLPs contain an extracellular domain and a short cytoplasmic 

tail without a kinase domain (Fig. 1.4) (Wang et al., 2018; Zipfel, 2014). Lacking an 

intracellular kinase domain, the majority of RLPs must interact constitutively with the 

adaptor kinase Suppressor of BAK1-Interacting Receptor-like Kinase 1 (SOBIR1) (Gust & 

Felix, 2014), and then recruit the Brassinosteroid Insensitive 1-Associated Kinase 

1/Somatic Embryogenesis Receptor Kinase 3 (BAK1/SERK3) for immune signaling upon 

recognition of the IP. For example, the tomato RLP Cf-4 is constitutively associated with 

SOBIR1 to provide stability and perceive Avr4, an Avr effector protein from C. fulvum, 

and then SERK1 and BAK1/SERK3 associate to induce an HR (Postma et al., 2016). In the 

same way, the tomato RLP Ve1, which recognizes Ave1, an effector protein from the 

Sordariomycete Verticillium dahliae, depends on SERK1 and BAK1/SERK3 to activate an 

HR (Fradin et al., 2011; Fradin et al., 2009). Receptor-like cytoplasmic kinases (RLCKs) 

associate with RLK complexes and trigger intracellular signaling through mitogen-

activated protein kinase (MAPK) cascades that, upon phosphorylation of different 

substrates, regulate plant immunity (Fig. 1.4) (Xu & Zhang, 2015). RLKs and RLPs are 

classified according to their ectodomain, with the leucine-rich repeat (LRR) ectodomain, 

which recognises peptides or proteins, being the largest subfamily (He et al., 2018). A 

well characterized LRR-RLK is the Flagellin-Sensing 2 (FLS2) that perceives the bacterial 

elicitor flagellin (fgl22) (Gómez-Gómez & Boller, 2000). There are other examples of 

extracellular domains in IPRs, such as the lysin motifs (LysM) that sense chitin and 

bacterial peptidoglycans, and the lectin-S domains that recognize bacterial 

lipopolysaccharide (LPS) (Ranf et al., 2015; Wan et al., 2012). 

 



Chapter 1: General introduction 
 
 

10 
 

 

Figure 1.4. Extracellular invasion patter receptor (IPR) activation and subsequent signal 

transduction in plants. Plasma membrane-localized receptor-like kinase (RLK) and receptor-like 

protein (RLP) invasion pattern (IP) receptors (IPRs), recognize IPs (shown as stars) of invading 

pathogens. At the basic level, RLKs contain an extracellular ectodomain (ED), a transmembrane 

domain (TM), and a cytoplasmic kinase domain (KD). Upon binding of an IP to a RLK receptor, 

hetero-dimerization of the receptor with a regulatory RLK is induced, which leads to 

phosphorylation and activation of both RLKs. On the contrary, RLPs lack the kinase domain often 

associated with RLKs. The activation of an RLP-RLK complex usually requires a ligand or IP-

dependent interaction with another RLK. Receptor-like cytoplasmic kinases (RLCKs) associate 

with RLK complexes to initiate mitogen-activated protein kinase (MAPK) cascades that further 

phosphorylate (P) diverse protein substrates to regulate not only plant immunity, but also other 

processes such as plant growth and development. Figure from He et al. (2018). 

 

1.2.2 Intracellular IPRs 

Intracellular IPRs frequently consist of a non-conserved amino (N)-terminal 

domain, central nucleotide-binding and oligomerization site (NB) and a carboxyl (C)-

terminal leucine-rich repeat (LRR) (Ma et al., 2020). These proteins, called NLRs, are 

classified depending on their N-terminal domain whether a Toll/interleukin 1 receptor 

(TIR), coiled-coil (CC) or (RPW8)-like coiled-coil domain (Martin et al., 2020; Petit-

Houdenot & Fudal, 2017; Takken & Goverse, 2012). NLR proteins are not designated to 

a single location in the cell, but have been found to localize to the cytoplasm, nuclear 

compartments, plasma membrane, and endomembranes (Heidrich et al., 2012; Kapos 

et al., 2019). Different effector detection strategies by NLR receptors have been shown. 



Chapter 1: General introduction 
 
 

11 
 

Direct recognition can occur when the effector interacts directly with the NLR, activating 

a defence response (Fig. 1.5A) (Dodds et al., 2006). Indirect recognition can be described 

by the Guard Model which proposes that the effector interacts with targets guarded by 

the NLR (Fig. 1.5B)  (Dangl & Jones, 2001). A third model, the Integrated Decoy Model, 

proposes that the NLR possesses an extra integrated domain (ID) that acts as a functional 

effector target that recognizes the effector indirectly (Fig. 1.5C) (Baggs et al., 2017). 

Direct or indirect effector recognition by NLRs trigger defence responses that generally 

give rise to an HR (Jones et al., 2016).  

Recently, the activation mechanisms of CC-NLRs and TIR-NLRs upon effector 

recognition have been described (Ma et al., 2020; Martin et al., 2020; Wang et al., 2019). 

Wang et al. (2019) studied the activation of the CC-NLR ZAR1 (HopZ-Activated Resistance 

1) from Arabidopsis by the AvrAC effector from the bacterium Xanthomonas campestris 

pv. campestris. ZAR1 in an inactive state is in a complex with the RLCK RKS1. Upon 

uridylation of the PBS1-like protein 2 (PBL2) by AvrAC, PBL2 activates RKS1 to induce 

conformational changes in ZAR1 to bind dATP or ATP which further facilitates structural 

remodelling of ZAR1. This structural remodeling results in the formation of a pentameric 

ZAR1-RKS1-PBL2 complex, also called a resistosome (Fig. 1.6A-B) (Wang et al., 2019). 

This funnel-shaped complex promotes ZAR1 integration into the plasma membrane to 

induce an immune response (i.e., HR-associated cell death) by disturbing the integrity of 

the plasma membrane or ionic homeostasis (Wang et al., 2019).  

Martin et al. (2020) studied the activation of the TIR-NLR ROQ1 (Recognition of 

XopQ1) from Nicotiana benthamiana by the effector XopQ (Xanthomonas outer protein 

Q) from Xanthomonas euvesicatoria. Upon physical interaction and recognition of XopQ, 

the LRR and post-LRR domains form a special ‘horseshoe-shape’ scaffold that encloses 

the effector and inhibits its ligand-binding function. Such recognition and activation lead 

to tetramerization of ROQ1 through the NB-ARC domains, bringing the TIR domains 

closer to form a resistosome (Fig. 1.6C). Such oligomerization exposes the NADase active 

site, in which the subsequent NAD+ cleavage leads to the release of adenosine 

diphosphate ribose (ADPR) that modulates cytosolic Ca2+ influx leading to cell death 

(Martin et al., 2020).  



Chapter 1: General introduction 
 
 

12 
 

 

Figure 1.5. Intracellular receptors (NLRs) recognize pathogen effector proteins through 

different strategies. Inactive NLRs bind adenosine diphosphate (ADP). Upon effector 

recognition, NLRs change to an activated conformation by binding adenosine triphosphate 

(ATP). Examples appear below each figure. (A) Direct effector recognition by interaction with 

the leucine-rich repeat (LRR) domain or the amino (N)-terminus of the NLR. Direct recognition 

of the ATR1 (Arabidopsis thaliana Recognized 1) effector from Hyaloperonospora arabidopsidis, 

by the C-terminal leucine-rich repeat (LRR) domain of the RPP1 (Recognition of Peronospora 

parasitica 1). Similarly, the Pi-ta NLR protein from rice recognizes directly the Avr-Pita effector 

from Magnaporthe oryzae. Direct recognition of the AvrL567 effector from Melampsora lini by 



Chapter 1: General introduction 
 
 

13 
 

the Toll/interleukin 1 receptor (TIR) domain from the L NLR protein of flax. (B) Indirect effector 

recognition by the NLR upon modification of the plant guardee or decoy by the effector. The 

tomato NLR protein Prf forms a complex with the intracellular kinase Pto. Pto interacts with the 

bacterial effector AvrPto from Pseudomonas syringae, activating the NLR and leading to an 

immune response. In a similar way, the A. thaliana NLR protein RPS2 detects the P. syringae 

effector AvrRpt2 recognition via the guardee protein RIN4 to trigger plant defences. (C) Indirect 

effector recognition by the NLR via integrated decoys often functioning in pairs with canonical 

NLRs. The Arabidopsis R protein pair RPS4 and RRS1 respond to the bacterial effectors AvrRps4 

and PopP2 (from P. syringae and Ralstonia solanacearum, respectively), via an integrated decoy 

at the C-terminus of RRS1 to trigger a defence response. Figure from Sun et al. (2020).   

 

 

 

Figure 1.6. Coiled-Coil (CC) and Toll-Interleukin 1 (TIR) nucleotide-binding site (NB) and 

leucine-rich repeat (LRR) receptor (NLR) oligomerize to form a resistosome. (A) Overall 

structure of the ZAR1 NLR from Arabidopsis. CC, beige; NB-ARC, pink, purple and light blue; LRR, 

blue. (B) Structure of ZAR1 resistosome and association with or integration into the plasma 

membrane. CC, beige; NB-ARC, pink, purple and light blue; LRR, blue; RKS1, yellow and PBL2 

green. (C) Overall structure of the ROQ1 (Nicotiana benthamiana NLR)-XopQ (Xanthomonas 

euvesicatoria effector) complex. TIR, yellow; NB-ARC, light to dark green; LRR, violet; post-LRR 

domain, light blue and XopQ, salmon. Figures adapted from Wang et al. (2019) and Martin et al. 

(2020).  

 

 



Chapter 1: General introduction 
 
 

14 
 

1.3 Features and functions of fungal effector proteins 

While fungal effectors can be secondary metabolites or small RNAs (Rodriguez-

Moreno et al., 2018), the majority of fungal effectors identified to date are 

proteinaceous. Typically, these proteinaceous effectors are small in size (<300 amino 

acid [aa] residues in length), highly expressed in planta, and contain an even number of 

cysteine residues involved in disulphide bond formation for protection against plant 

proteases (Dalio et al., 2018; Mesarich et al., 2018). They also often possess an amino 

N-terminal signal peptide for secretion to the host environment (Mesarich et al., 2014), 

but tend to lack a transmembrane domain (TM) for integration into the fungal plasma 

membrane, or glycophosphatidylinositol (GPI) anchor modification site for attachment 

to the fungal plasma membrane or cell wall. Nevertheless, there are examples of 

effector proteins that do not possess these common characteristics, such as Vdlsc1, an 

unconventionally secreted effector protein from V. dahliae, which lacks a signal peptide 

(Liu et al., 2014), or VPS9, a conventionally secreted effector protein from the 

Basidiomycota fungus Puccinia graminis f. sp. tritici that is 744 aa in size (Nirmala et al., 

2011).  

Some effector proteins are race-specific, while others are highly conserved across 

fungi, also known as ʻʻcore effectorsʼʼ (Stergiopoulos et al., 2010; Wang & Wang, 2018). 

Necrosis and ethylene-inducing peptide 1 (Nep1)-like proteins (NLPs), for example, are 

conserved effector proteins secreted by fungal species with necrotrophic lifestyles to 

trigger tissue necrosis for successful colonization (Bashi et al., 2010; Bohm et al., 2014). 

These include SsNep1 and SsNep2 from Sclerotinia sclerotiorum, which induce cell death 

when transiently expressed in tobacco leaves (Bashi et al., 2010), and both MpNep1 and 

MpNep2 from Moniliophthora perniciosa, which induce necrosis and ethylene emission 

in tobacco and cacao leaves (Garcia et al., 2007). Avr4 and Ecp2 from C. fulvum are 

examples of core effectors with functional orthologs widely distributed among other 

Dothideomycete species. Effectors can act in the plant apoplast (apoplastic effectors) or 

inside plant cells (cytoplasmic effectors). Some examples are detailed below. 

 



Chapter 1: General introduction 
 
 

15 
 

1.3.1 Apoplastic effector proteins  

The apoplast is the narrow space between the pathogen cell wall and the plant plasma 

membrane (Rocafort et al., 2020; Wang et al., 2020) that offers a nutrient supply for the 

pathogen to proliferate. However, this narrow space is also armed with plant proteases and 

hydrolases that interfere with pathogen infection. In response, fungal pathogens deploy 

apoplastic effectors to protect their cell walls against hydrolysis by host chitinase enzymes, 

or to sequester fungal cell wall oligosaccharides that are released by plant hydrolases to 

prevent their recognition as PAMPs by PRRs. An example is Avr4 from C. fulvum, which 

protects against plant chitinases by binding to chitin in the fungal cell wall (van den Burg et 

al., 2006). Avr4 homologs have been identified in other Dothideomycete plant pathogens 

such as Dothistroma septosporum, Pseudocercospora fijiensis, Cercospora apii, Cercospora 

beticola and Cercospora nicotianae (de Wit et al., 2012; Stergiopoulos et al., 2010). Other 

examples include the LysM domain effectors, which sequester chitin oligosaccharides to 

prevent their recognition by host LysM IPRs (de Jonge & Thomma, 2009; Kombrink et al., 

2011; Mentlak et al., 2012). Slp1 from M. oryzae, for instance, sequesters chitin-

oligosaccharides in order to suppress chitin-induced plant immune responses (Mentlak et 

al., 2012).  

During colonization of the apoplast, plant-pathogenic fungi also need to overcome 

defences mediated by plant proteases. Inhibition of these enzymes is one of the 

strategies used by these organisms to overcome plant defence. For example, the 

biotrophic fungus Ustilago maydis inhibits maize cysteine proteases by secreting the 

effector Pit2, which is essential for fungal virulence (Mueller et al., 2013). In addition, 

the same fungus secretes the effector Pep1 (Protein essential during penetration 1) to 

inhibit maize peroxidases and thus interfere with the oxidative burst that generates ROS 

as a defence response (Hemetsberger et al., 2012). Another barrier to colonizing the 

apoplast is the presence of anti-microbial compounds. Therefore fungal pathogens 

secrete enzymes as a strategy for detoxification of these compounds. For example,               

C. fulvum secretes CfTom1, a glycosyl hydrolase that hydrolyses α-tomatine (a major 

saponin of tomato) into the non-toxic tomatidine (Ökmen et al., 2013).  



Chapter 1: General introduction 
 
 

16 
 

1.3.2 Cytoplasmic effector proteins  

Cytoplasmic effectors are translocated inside host cells where they modulate plant 

immunity and physiology. Once inside the cells they can affect processes such as 

hormone signaling, secondary metabolite synthesis, transcription systems, and can 

inactivate important components in PRRs, hence disturbing MAPK cascades (Asai & 

Shirasu, 2015; Liu et al., 2014; Lo Presti et al., 2015). As mentioned, there are effectors 

that influence hormone signaling by manipulating, for example, salicylic acid (SA) 

production. Such is the case of the V. dahliae effector Vdlsc1 that hydrolyses 

isochorismate, the precursor of SA, to suppress its accumulation in the host (Liu et al., 

2014). Similarly, the chorismate mutase effector protein Cmu1 is delivered into host 

cells by U. maydis to reduce the production of SA (Djamei et al., 2011). Another example 

is AvrLm4-7 from L. maculans that also manipulates the signaling pathways involved in 

the biosynthesis of SA and ethylene (Nováková et al., 2016). Unlike other fungal 

pathogens such as rusts and powdery mildews that produce haustoria likely involved in 

effector translocation, the effector delivery mechanism into plant cells by filamentous 

pathogens that are restricted to the apoplast remains unknown. Interestingly, plant cell-

to-cell movement of cytoplasmic effectors has been reported. The F. oxysporum 

effectors Avr2 and Six5 interact at and manipulate plasmodesmata to facilitate cell-to-

cell movement of Avr2 (Cao et al., 2018).  

1.3.3 Avirulence effector proteins 

As mentioned in section 1.1, effector proteins of plant-pathogenic fungi can 

sometimes be recognized as Avr effectors by plant R proteins to trigger IPTRs. Such Avr–

R recognition events can help to identify new R genes to incorporate into new cultivars, 

as well as to understand how fungal pathogens cause disease. Hence, it is fundamental 

to identify new Avr effector proteins as a base to improve future plant disease control 

strategies (Zhang & Coaker, 2017). Many Avr effector proteins have been identified from 

plant-pathogenic fungi over recent years using different approaches, including 

proteomics or reverse genetics, map-based cloning, homology-based searches, 

association mapping, comparative genomics and transcriptomics (Kanja & Hammond-

Kosack, 2020).  



Chapter 1: General introduction 
 
 

17 
 

The C. fulvum avirulence effectors Ecp1, Ecp2-1, Ecp4, Ecp5, Ecp6, Avr4, Avr4E and 

Avr9 were identified using reverse genetics (Joosten et al., 1994; Joosten & de Wit, 1988; 

Laugé. et al., 2000; Schottens-Toma & de Wit, 1988; Van den Ackerveken, Van Kan, et 

al., 1993; van Kan et al., 1991; Westerink et al., 2004; Wubben et al., 1994). Avr2 was 

identified using an effectoromics approach (Luderer et al., 2002), and Avr5 was 

identified through a bioinformatics and transcriptome sequencing approach (Mesarich 

et al., 2014). The avirulence effector AvrStb6 from Z. tritici was identified using a 

genome-wide association study (GWAS) in combination with quantitative trait locus 

(QTL) mapping (Zhong et al., 2017). AvrLm1, AvrLm6, AvrLm4-7, AvrLm5-9, AvrLm10, 

AvrLm11 and AvrLm14 from L. maculans were identified using a map-based cloning 

approach (Balesdent et al., 2013; Degrave et al., 2021; Fudal et al., 2007; Gout et al., 

2006; Parlange et al., 2009; Petit-Houdenot et al., 2019; Van de Wouw et al., 2014). 

AvrLm2 was identified through comparative genomics (Ghanbarnia et al., 2015), and the 

laborious identification of AvrLm3 included genetic mapping, RNA-seq, next generation 

sequencing (NGS) of bacterial artificial chromosome (BAC) clones and de novo assembly 

approaches (Plissonneau et al., 2016). Characterized Avr effector proteins identified 

from Dothideomycete plant-pathogenic fungi, and their cognate R proteins, are 

summarized in Table 1.1. 

 

 

 

 

 

 

 

 



Chapter 1: General introduction 
 
 

18 
 

 

Table 1.1. Identified Avirulence (Avr) effector proteins from plant-pathogenic 
Dothideomycete fungi and their corresponding plant resistance (R) proteins. 

Plant 
pathogen 

Disease  Avirulence 
effector 
protein 

Size 
(aa)a 

Cys  
No.b 

Corresponding 
R proteinc 

Reference 

Leptosphaeria 
maculans 

Black leg 
disease 
of 
Brassica 
crops 

AvrLm1 205 1 LepR3 (RLP) (Gout et al., 
2006; Ma & 
Borhan, 
2015) 

AvrLm2 232 8 Rlm2 (RLP) (Ghanbarnia 
et al., 2015; 
Larkan et al., 
2015) 

AvrLm3 160 10 Rlm3 (Plissonneau 
et al., 2016) 

AvrLm4-7 143 8 Rlm4 and 
Rlm7 (WAKL) 

(Haddadi et 
al., 2021; 
Parlange et 
al., 2009) 

AvrLm5-9 141 6 Rlm9 and 
Rlm5 (WAKL) 

(Ghanbarnia 
et al., 2018; 
Larkan et al., 
2020; 
Plissonneau 
et al., 2018; 
Van de 
Wouw et al., 
2014) 

AvrLm6 144 6 Rlm6 (Fudal et al., 
2007) 

AvrLm10A     
AvrLm10B 

120                  
178  

7                            
1 

Rlm10 (Petit-
Houdenot et 
al., 2019) 

AvrLm11 95 1 Rlm11 (Balesdent 
et al., 2013) 

AvrLm14 134 4 Rlm14 (Degrave et 
al., 2021) 

Zymoseptoria 
tritici  

Septoria 
tritici 
blotch of 
wheat 

AvrStb6 82 12 Stb6 (RLK) (Saintenac et 
al., 2018; 
Zhong et al., 
2017) 

Avr3D1 92 8 Stb7 (Meile et al., 
2018) 

 

 

 



Chapter 1: General introduction 
 
 

19 
 

Table 1.1. Continued.  

Plant 
pathogen 

Disease  Avirulence 
effector 
protein 

Size 
(aa)a 

Cys  
No.b 

Corresponding 
R proteinc 

Reference 

Cladosporium 
fulvum 

Leaf 
mould of 
tomato 

Ecp1 96 8 Cf-Ecp1 (Laugé et al., 
1997) 

Ecp2-1 165 4 Cf-Ecp2 (Laugé et al., 
1997) 

Ecp4 119 6 Cf-Ecp4 (Laugé. et 
al., 2000) 

Ecp5 115 5 Cf-Ecp5 (Laugé et al., 
2000) 

Ecp6 222 8 Cf-Ecp6 (Bolton et 
al., 2008) 

Avr2 78 8 Cf-2.1 and     
Cf-2.2 (RLP) 

(Dixon et al., 
1996; 
Luderer et 
al., 2002; 
Rooney, 
2005) 

Avr4 135 8 Cf-4 (RLP) (Joosten et 
al., 1994; 
Thomas et 
al., 1997)  

Avr4E 121 6 Cf-4E (RLP) (Takken et 
al., 1999; 
Westerink et 
al., 2004) 

Avr5 103 10 Cf-5 (RLP) (Mesarich et 
al., 2014; 
Dixon et al., 
1998) 

Avr9 63 6 Cf-9 (RLP) (Jones et al., 
1994; van 
Kan et al., 
1991) 

aLength in amino acids (aa), including signal peptide. 
bCysteine number in mature protein. 
cRLP, receptor-like protein; RLK, receptor-like kinase; WAKL wall associated kinase-like. 

 

 

 

 



Chapter 1: General introduction 
 
 

20 
 

1.4 The New Zealand horticulture industry 

In 2020, the total value of the New Zealand horticultural industry was estimated 

to exceed $10 billion, including a record high of $6.65 billion worth of exports 

(http://freshfacts.co.nz, issue 2020). The export value has increased more than 60% in 

the past 10 years thanks to the investment from research to post-harvest practices. 

Apple and tomato are two crops produced in New Zealand for local and international 

consumption, contributing greatly to its economy. Apple, for instance, was the second 

largest horticultural export of New Zealand in 2020, in the fresh fruit category. More 

specifically, in the same year, New Zealand exported 402,000 tonnes of apple, reaching 

a record value of $876 million. Apple statistics show a stable growth of the apple fruit 

industry year-on-year (Table 1.2), which is anticipated to reach a value of 1 billion by 

2022. 

In total, 125 tomato greenhouses, with a planted area of 120 ha are present in 

New Zealand. The domestic sales of tomato reached a value of $120 million in 2020, and 

a value of $11.2 million for export the same year (http://freshfacts.co.nz, issue 2020) 

(Table 1.3). 

 

Table 1.2. New Zealand apple industry overview. Source: FreshFacts, issue 2020 

(http://freshfacts.co.nz) 

Year 2005 2010 2015 2018 2019 2020 

National export production             

(‘000 tonnes) 

 315 260 331 377 395 402 

Exports ($ million, fob1) 387 324.6 561.8 732.9 828.8 876.3 

Area planted (ha2) 10,764 8,630 8,566 9,139 10,179 10,396 

1Exports given as free on-board (fob) values. 
2ha; hectares. 

 

 

http://freshfacts.co.nz/
http://freshfacts.co.nz/
http://freshfacts.co.nz/


Chapter 1: General introduction 
 
 

21 
 

Table 1.3. New Zealand tomato industry overview. Source: FreshFacts, issue 2020 

(http://freshfacts.co.nz). 

Year 2005 2010 2015 2018 2019 2020 

Total crop volume1  

 (tonnes) 

40,000 40,000 42,400 42,400 n/a 42,000 

Exports ($ million, fob2) 8.5 10.1 8.1 9.6 11.2 11.2 

Domestic sales ($ 

million, fob) 

105 108 91.2 200 176 120 

1Greenhouse production. 
2Exports given as free on-board (fob) values. 
n/a: not available 

 

Climate change has brought many challenges for apple growers such as colour 

development (red color reduction), sunburn and increased pests and diseases 

(FreshFacts NZ, issue 2019). Regarding tomato greenhouse production, it is less 

influenced by climatic factors. However, with increasing rainfall and humidity in some 

regions, traditional farming of tomatoes in the open field could also be at risk (Silva et 

al., 2017).  Biotic factors, such as pathogens that cause disease, are a major threat for 

apple and tomato production. Many of these diseases are caused by Dothideomycete 

fungi. Two prime examples are apple scab disease caused by V. inaequalis and tomato 

leaf mould disease caused by C. fulvum. Future challenges in the horticulture industry of 

New Zealand include responding to current and future plant disease outbreaks, in order 

to meet the qualitative and quantitative demands of the industry. Research priorities 

must include plant-pathogen interactions to provide knowledge for the development of 

long-lasting resistant plant varieties. The focus of this thesis is these two important 

pathogens to better understand the molecular mechanisms they use to invade their 

respective hosts, as well as the molecular mechanisms their hosts use to detect them.  

 

 

http://freshfacts.co.nz/


Chapter 1: General introduction 
 
 

22 
 

1.5 Cladosporium fulvum 

Tomato (Solanum lycopersicum) leaf mould is caused by the asexual, non-obligate 

biotrophic Dothideomycete fungus C. fulvum (syn. Passalora fulva and Fulvia fulva) 

(Braun et al., 2003; Thomma et al., 2005). C. fulvum affects tomato plants worldwide, 

but it is in greenhouses and high-tunnel environments where the main problem persists 

due to the favorable conditions, such as moderate temperatures and a high relative 

humidity  (Mesarich et al., 2018).  This fungal disease was first described by Cooke (1883) 

and its origin is presumably in South America, where the provenance of cultivated 

tomato has been corroborated (Jenkins, 1948).  

1.5.1 Infection cycle of C. fulvum 

Infection starts when conidia, with the aid of wind or water, spread and land on the 

abaxial side of a tomato leaf. When high relative humidity conditions are present, 

conidia germinate and form a thin runner hypha that grows over the leaf surface  (Bond, 

1938; De Wit, 1977; Lazarovits & Higgins, 1976). Then, the main germ tube or a lateral 

hyphal branch is able to enter the leaf when an open stoma is encountered. After the 

inner leaf is reached, the hypha enlarges and continues growing between the mesophyll 

cells (Fig. 1.7C), forming long and branched hyphal structures that form hyphal 

aggregations in the substomatal spaces approximately 10-14 days after the initial 

infection. After aerial mycelia are formed, conidiophores are released to the exterior 

through stomata, where they produce conidia, which are subsequently dispersed to 

spread the disease (Fig. 1.7D) (de Wit et al., 2012; Thomma et al., 2005). The disease 

symptoms are characterized by pale green to yellow spots on the adaxial leaf surface 

that turn brown after sporulation (Fig. 1.7 A-B), leaf wilting, and partial defoliation that 

in severe infections can lead to plant death (Thomma et al., 2005). Besides affecting 

leaves, C. fulvum can also infect stems, flowers and fruits (Butler & Jones, 1949).  



Chapter 1: General introduction 
 
 

23 
 

 

Figure 1.7.  Tomato leaf mould disease symptoms and development. (A) Cladosporium fulvum 

sporulating on the abaxial side of a tomato (Solanum lycopersicum) plant two weeks-post 

inoculation; (B) C. fulvum sporulating on the abaxial side of a leaflet at two weeks post-

inoculation; (C) C. fulvum (transgenic strain expressing green fluorescent protein; GFP) runner 

hyphae at the surface of the leaf and penetrating a stoma of tomato at four days post-

inoculation. (D) C. fulvum (GFP-transgenic strain) conidiophores emerging from a stoma at 10 

days post-inoculation. Figure adapted from de Wit et al. (2012). 

 

1.5.2 Leaf mould control methods 

Leaf mould disease is controlled in different ways. Cultural methods rely on keeping 

the relative humidity in greenhouses below 85%, providing enough ventilation and light, 

adequate spacing between rows and plants, and avoiding persistent moisture on leaves. 

Additionally, it is important to reduce the primary inoculum by removing and destroying 

remaining plant debris. Along with the mentioned cultural practices, fungicide spray 

programmes are used to help control the disease (Veloukas et al., 2007). Among these 

are the demethylation inhibitors (DMIs) which bind to the heme iron of the cytochrome 

P450 sterol 14α-demethylase (Cyp51) to inhibit sterol biosynthesis in the fungal 

membranes (Yuzo & Yuri, 1987). As observed in other fungal pathogens, resistance to 

this type of fungicide could be the result of mutations or overexpression of the CYP51A1 

gene in C. fulvum (Cordero-Limon et al., 2021; Köller, 1992; Ma et al., 2006; Villani et al., 

2016; Wang et al., 2015). A specific type of DMI, the triazoles, are effective fungicides 

against C. fulvum (Wang et al., 2017). So far, no resistance to triazoles have been 

reported in C. fulvum. Another class of fungicides used are the methyl benzimidazole 



Chapter 1: General introduction 
 
 

24 
 

carbamate (MBC) fungicides that bind the β-tubulin in order to prevent fungal mitosis 

and cell division (Ma & Michailides, 2005). C. fulvum isolates resistant to MBC fungicides 

have been reported through point mutation at codons 198 and 200, leading to Glu-to-

Ala and Phe-to-Tyr amino acid substitutions, respectively, in the β-tubulin protein (Yan 

et al., 2008). Finally, strobirulin or quinone outside inhibitors (QoI), which unpair the 

ability of the pathogen to produce energy through inhibition of mitochondrial 

respiration (i.e. binding to the cytochrome bc1 [CYTB] enzyme complex at the outer 

quinone oxidizing site [QoI site] (Gisi et al., 2002). However, resistance to QoI fungicides 

in C. fulvum has been reported, and results from a Phe-to-Leu amino acid substitution 

at position 129 of CYT B (Watanabe et al., 2017). In addition to cultural and fungicide 

control methods, cultivars of tomato with R genes active against C. fulvum are used.  

1.5.3 Molecular aspects of the C. fulvum-tomato interaction 

The C. fulvum-tomato interaction has been used as a pathosystem model for 

understanding gene-for-gene interactions between pathogen Avr effector genes and 

host plant R genes. The products of tomato R genes, designated as (Cf) for resistance to 

Cladosporium fulvum, mediate recognition of corresponding Avr effector gene products 

from C. fulvum (van den Burg et al., 2006). When the matching Cf and Avr genes are 

present, a resistance response is triggered in the plant; in this manner the fungus is 

avirulent and can no longer infect the host (incompatible interaction). If no matching Cf 

and Avr genes are present, then there is no recognition by the plant, and thus the fungus 

is virulent and can successfully colonize the host (compatible interaction) (de Wit et al., 

1997).  

1.5.3.1 Compatible interaction 

Under optimum relative humidity and temperature conditions, conidia germinate 

and subsequently differentiate runner hyphae. Then, runner hyphae enter tomato 

leaves through open stomata (Joosten & de Wit, 1999). C. fulvum does not form 

haustoria to feed off the plant. Instead, it remains in the leaf apoplast in order to obtain 

nutrients such as sugars and amino acids, where it secretes effector proteins to interfere 

with plant defence responses and promote colonization (Joosten et al., 1990). These 

include at least 75 small secreted proteins (SSPs) of less than 300 aa residues in length 

(Mesarich et al., 2018), most of which have an even number of cysteines residues that 



Chapter 1: General introduction 
 
 

25 
 

form disulphide bridges that provide stability in the harsh protease-rich tomato 

apoplast. Effector proteins of C. fulvum can be divided into extracellular proteins (ECPs) 

produced by all strains, and avirulence proteins (Avrs) that are race-specific (Rooney, 

2005; van Esse et al., 2008). As mentioned in Section 1.5.1, approximately 14 days after 

infection, hyphal aggregations form in the substomatal spaces and release conidia 

through stomata leads to spread of the disease (Thomma et al., 2005). A compatible 

interaction is illustrated in (Fig. 1.11A) (Ökmen, 2013).  

1.5.3.2 Incompatible interaction 

Regarding conidial germination, runner hyphae differentiation and stomatal 

penetration, there is no difference between a compatible and incompatible interaction. 

It is when the runner hyphae enter the apoplast that host defence responses are 

activated to halt the growth of the fungus. These responses involve collapse of the host 

mesophyll cells that are proximate to the fungal hyphal cells and callose formation (De 

Wit, 1977). At the molecular level, there is a fast accumulation of PR proteins such as           

β-1,3-glucanases, chitinases and cysteine proteases (Wubben et al., 1993). However, the 

most efficient outcome of the incompatible interaction is the HR, already described in 

Section 1.1 which halts the growth of the fungus (de Wit et al., 2009). An incompatible 

interaction is illustrated in (Fig. 1.11B) (Ökmen, 2013). 

1.5.4 Cladosporium fulvum effectors 

A large number of C. fulvum effectors have been identified using different 

approaches (Section 1.3.3). From these, only Avr2, Avr4, Ecp6 and CfTom1 have been 

functionally characterized.  

1.5.4.1 Characterized C. fulvum effectors 

Avr2 is a pre-protein of 78 aa in length with a 20 aa predicted signal sequence and 

contains eight cysteine residues. Avr2 inhibits diverse tomato cysteine proteases 

including Rcr3 (required for C. fulvum resistance) (Dixon et al., 2000; Kruger et al., 2002; 

Rooney, 2005), and the close relative Pip1 (Phytophthora-inhibited protease 1) (van Esse 

et al., 2008). Heterologous expression of Avr2 in Arabidopsis thaliana enhances 

susceptibility to Botrytis cinerea and V. dahliae. In the same way, tomato lines 

expressing Avr2 increase susceptibility to race 2 C. fulvum strains (lacking Avr2), showing 



Chapter 1: General introduction 
 
 

26 
 

that Avr2 is required for virulence (van Esse et al., 2008). Recently, it has been shown 

that Rcr3 homologs in tomato, potato, eggplant, pepper and petunia can be inhibited by 

Avr2; however, only Rcr3 homologs from tomato and potato can trigger an HR in the 

presence of Cf-2 and Avr2, illustrating the evolution of Rcr3 (Kourelis et al., 2020). Such 

Cf-2-dependent Avr2 recognition is correlated with the evolutionary distance to tomato; 

as the phylogenetic distance to tomato Rcr3 increases, the likelihood of a Cf-2-

dependent HR decreases (Kourelis et al., 2020).  

The mature Avr4 protein contains 86 aa with eight cysteine residues involved in 

disulphide bridge formation (Joosten et al., 1994). Avr4 protects the hyphae against 

plant chitinases by binding to the fungal cell walls and in doing so, prevents the release 

and recognition of chitin fragments by PRRs (van den Burg et al., 2006). The chitin- 

binding ability of Avr4 is mediated by the carbohydrate-binding module family 14 

(CBM14) domain (van den Burg et al., 2006). The solved crystal structure of CfAvr4 

revealed structural similarity to other CBM14 family members (Fig. 1.8A) (Hurlburt et 

al., 2018). In the same study, an effector-ligand co-crystallization of Avr4-chitohexaose 

[(GlcNAc)6] uncovered that two Avr4 molecules dimerize and form a sandwich structure 

by encapsulating two molecules of chitohexahose (GlcNAc)6 (Fig. 1.8B) (RSCB protein 

data bank (PDB) ID: 6BN0) (Hurlburt et al., 2018).  

The role of Avr4 in virulence has been demonstrated (van Esse et al., 2007); 

heterologous expression of Avr4 in A. thaliana increased susceptibility to B. cinerea and 

Plectosphaerella cucumerina. In addition, the silencing of this effector resulted in less 

colonization and biomass production by Avr4-silenced C. fulvum strains on tomato 

leaves (van Esse et al., 2007). Avr4 homologues have been identified in other 

Dothideomycete fungi such as P. fijiensis and Cercospora species (Stergiopoulos et al., 

2010). The M. fijiensis Avr4 is a functional ortholog that protects fungi against plant 

chitinases in the same way as C. fulvum Avr4 and induces a strong Cf-4-dependent HR 

(Stergiopoulos et al., 2010). Interestingly, a recently described paralog of Avr4 from 

Pseudocercospora fuligena, PfAvr4-2, was found to contain a non-functional chitin-

binding domain. Instead, this paralog interacts with pectin in plant cell walls and 

interferes with Ca2+-mediated cross-linking at the cell-cell junction zones. In this manner, 

it affects the structure of the plant cell wall and makes it more accessible for enzymatic 

degradation (Chen et al., 2021).  



Chapter 1: General introduction 
 
 

27 
 

 

Figure 1.8. Crystal structure of the Avr4 effector protein from Cladosporium fulvum. (RSCB 

protein data bank (PDB) ID:  6BN0) (A) The Avr4 monomer is composed of an amino (N)-terminal 

α-helix (H1), an unorganized β-sandwich fold with three anti-parallel β-strands (A1-A3), two anti-

parallel β-strands (B4 and B5) forming a small β-sheet, and a carboxyl (C)-terminal α-helix (H2). 

Avr4 is bound to a chitohexaose (GlcNAc)6 molecule (shown in sticks). (B) The CfAvr4-(GlcNAc)6 

dimeric structure is composed by two Avr4 monomers (shown in purple and blue), binding to 

two molecules of (GlcNAc)6 (shown in sticks). Disulfide bonds in both structures are shown in 

yellow. Figures adapted from Hurlburt et al. (2018). 

 

Ecp6 is a well characterized protein of 222 amino acids, with a signal peptide of 

18 amino acids, eight cysteines and contains three LysM carbohydrate binding domains 

(LysM1-LysM3) (Fig. 1.9) (RSCB protein data bank (PDB) ID: 4B8V) (Bolton et al., 2008; 

Sanchez-Vallet et al., 2013). Unlike Avr4 that prevents hydrolysis of fungal cell walls, 

Ecp6 sequesters chitin fragments released by plant chitinases and thus prevents the 

activation of defence responses (de Jonge et al., 2010). More specifically, the binding of 

chitin is mainly enabled by the dimerization of domains LysM1 and LysM3 to form a 

chitin-binding groove which outcompetes host PRRs for the binding of chitin (de Jonge 

et al., 2010; Sanchez-Vallet et al., 2013). Silencing of the Ecp6 gene demonstrated its 

role in virulence, with silenced transformants having less biomass in planta (Bolton et 

al., 2008). Interestingly, Ecp6 orthologs are not only present in other Dothideomycetes 

such as L. maculans, Z. tritici and P. fijiensis, but also in the Sordariomycete M. oryzae, 

and the Leotiomycetes Sclerotinia sclerotiorum and B. cinerea, indicating the importance 



Chapter 1: General introduction 
 
 

28 
 

of avoiding chitin-triggered plant defence responses across distinct classes of fungal 

pathogens (Bolton et al., 2008).  

 

Figure 1.9. Crystal structure of the Ecp6 effector protein from Cladosporium fulvum. (RSCB 

protein data bank (PDB) ID: 4B8V). Structure of an Ecp6 dimer (left monomer in orange) and 

three LysM domains (in three shades of blue) of the right monomer with flexible loop (grey) 

between LysM1 and LysM2. The chitin tetramer and the four disulfide bridges are indicated in 

green and yellow sticks, respectively. In the right monomer the two chitin-binding loops are 

shown in red and green for each of the LysM domains. Figure from Sánchez-Vallet et al. (2013).  

 

CfTom1 is a secreted enzyme and part of glycosyl hydrolase 10 family (GH10). It 

contains 345 amino acids with a predicted secretion signal of 19 amino acids, and two 

cysteines. This effector degrades the fungitoxic α-tomatine into tomatidine, a less toxic 

metabolite. The requirement of CfTom1 for full virulence was demonstrated by a 

targeted gene deletion, where a reduction of fungal biomass of Δcftom mutants was 

observed at 10 dpi onwards (Ökmen et al., 2013).  

1.5.4.2 Uncharacterized C. fulvum effectors 

The Ecp1 gene encodes a precursor protein of 96 aa in size, which after the removal 

of its secretion signal and the activity of plant proteases results in a stable protein of 65 

aa with eight cysteine residues. Ecp2 has a 165 aa precursor protein that is processed 

similarly to Ecp1 and results in a protein of 142 aa with four cysteines (Joosten & de Wit, 

1988; Van den Ackerveken, Van Kan, et al., 1993; Wubben et al., 1994). Using a gene 



Chapter 1: General introduction 
 
 

29 
 

replacement approach, Ecp1 and Ecp2 were found to be invo