Smut fungi are a large group of biotrophic plant pathogens that infect mostly monocot species, including economically relevant cereal crops. For years,
Ustilago maydis
has stood out as the model ...system to study the genetics and cell biology of smut fungi as well as the pathogenic development of biotrophic plant pathogens. The identification and functional characterization of secreted effectors and their role in virulence have particularly been driven forward using the
U. maydis
-maize pathosystem. Today, advancing tools for additional smut fungi such as
Ustilago hordei
and
Sporisorium reilianum
, as well as an increasing number of available genome sequences, provide excellent opportunities to investigate in parallel the effector function and evolution associated with different lifestyles and host specificities. In addition, genome analyses revealed similarities in the genomic signature between pathogenic smuts and epiphytic
Pseudozyma
species. This review elaborates on how knowledge about fungal lifestyles, genome biology, and functional effector biology has helped in understanding the biology of this important group of fungal pathogens. We highlight the contribution of the
U. maydis
model system but also discuss the differences from other smut fungi, which raises the importance of comparative genomic and genetic analyses in future research.
The basidiomycete smut fungi are predominantly plant parasitic, causing severe losses in some crops. Most species feature a saprotrophic haploid yeast stage, and several smut fungi are only known ...from this stage, with some isolated from habitats without suitable hosts, e.g. from Antarctica. Thus, these species are generally believed to be apathogenic, but recent findings that some of these might have a plant pathogenic teleomorph counterpart cast doubts on the validity of this hypothesis. Here, four genomes of species previously assigned to the polyphyletic genus
Pseudozyma
were re-annotated and compared with published smut pathogens. It was found that 113 genes coding for putative secreted effector proteins were conserved among smut-causing and
Pseudozyma
genomes. Among these were several validated effector genes, including
Pep1
. Orthologs of this well-characterised effector from
Pseudozyma
yeasts were further analysed and checked for their ability to complement a
Pep1
-deficient mutants of
Ustilago maydis
. By genetic complementation, we show that
Pep1
homologs from the supposedly apathogenic yeasts restore virulence in
Pep1
-deficient mutants
Ustilago maydis
. Thus, it is concluded that
Pseudozyma
species have likely retained a suite of effectors, which hints at the possibility that
Pseudozyma
species have kept an unknown plant pathogenic stage for sexual recombination. However, it cannot be excluded that these effectors might also have positive effects also when colonising plant surfaces.
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EMUNI, FIS, FZAB, GEOZS, GIS, IJS, IMTLJ, KILJ, KISLJ, MFDPS, NLZOH, NUK, OBVAL, OILJ, PNG, SAZU, SBCE, SBJE, SBMB, SBNM, UKNU, UL, UM, UPUK, VKSCE, ZAGLJ
In this work, 29 pepper cultivars that represent the diversity of types and varieties grown in Turkey were analyzed for water-soluble antioxidant capacity and phenolic and vitamin C contents. In ...addition, 14 non-Turkish cultivars were tested for comparison. Significant diversity was observed in the different cultivars with the most variation (7.4-fold) seen for total antioxidant capacity, which ranged from 2.57 to 18.96 mmol Trolox/kg. Vitamin C content for the peppers ranged from 522 to 1631 mg·kg-1, a 3.1-fold difference, whereas total phenolic content for the pepper cultivars ranged from 607 to 2724 mg·kg-1, a 4.5-fold difference. When cultivars were grouped by morphology/use, it was found that some types had significantly more variation and higher antioxidant activities than other types. Thus, for water-soluble antioxidant capacity, most variation was seen in long, blunt-ended Çarliston types, whereas long, pointed Sivri peppers had the highest mean capacity. Bell-shaped Dolmalk and Sivri peppers had the most variation for phenolic content, but fancy Süs and Sivri types had the highest means for this trait. Dolmalk types showed the most variation for vitamin C content, whereas Süs and Sivri peppers had the highest means for this character. All three parameters were significantly and positively correlated with the strongest correlation between total antioxidant capacity and phenolic content (r = 0.71). The presence of significant variation for antioxidant content in Turkish germplasm indicates that this material can be used for improvement and genetic mapping of nutritional content in pepper.
Summary
Fungal
W
or1‐like proteins are conserved transcriptional regulators that are reported to regulate the virulence of several plant pathogenic fungi by affecting the expression of virulence ...genes. Here, we report the functional analysis of
CfWor1
, the homologue of
W
or1 in
C
ladosporium fulvum
. Δ
cfwor1
mutants produce sclerotium‐like structures and rough hyphae, which are covered with a black extracellular matrix. These mutants do not sporulate and are no longer virulent on tomato. A
CE
.
CfWor1
transformant that constitutively expresses
CfWor1
produces fewer spores with altered morphology and is also reduced in virulence.
RNA
‐seq and
RT‐qrtPCR
analyses suggest that reduced virulence of
Δ
cfwor1
mutants is due to global downregulation of transcription, translation and mitochondrial respiratory chain. The reduced virulence of the
CE
.
CfWor1
transformant is likely due to downregulation of effector genes. Complementation of a non‐virulent Δ
fosge1
(
W
or1‐homologue) mutant of
F
usarium oxysporum
f. sp.
lycopersici
with
CfWor1
restored expression of the
SIX
effector genes in this fungus, but not its virulence. Chimeric proteins of
CfWor1
/
FoSge
1 also only partially restored defects of the Δ
fosge1
mutant, suggesting that these transcriptional regulators have functionally diverged. Altogether, our results suggest that
CfWor1
primarily regulates development of
C
. fulvum
, which indirectly affects the expression of a subset of virulence genes.
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BFBNIB, FZAB, GIS, IJS, KILJ, NLZOH, NUK, OILJ, SAZU, SBCE, SBMB, UL, UM, UPUK
Summary
Previously, we have determined the nonhost‐mediated recognition of the
MfA
vr4 and
MfE
cp2 effector proteins from the banana pathogen
M
ycosphaerella fijiensis
in tomato, by the cognate
C
f‐4 ...and
C
f‐Ecp2 resistance proteins, respectively. These two resistance proteins could thus mediate resistance against
M
. fijiensis
if genetically transformed into banana (
M
usa
spp.). However, disease resistance controlled by single dominant genes can be overcome by mutated effector alleles, whose products are not recognized by the cognate resistance proteins. Here, we surveyed the allelic variation within the
MfA
vr4
,
MfE
cp2
,
MfE
cp2‐2
and
MfE
cp2‐3
effector genes of
M
. fijiensis
in a global population of the pathogen, and assayed its impact on recognition by the tomato
C
f‐4 and
C
f‐
E
cp2 resistance proteins, respectively. We identified a large number of polymorphisms that could reflect a co‐evolutionary arms race between host and pathogen. The analysis of nucleotide substitution patterns suggests that both positive selection and intragenic recombination have shaped the evolution of
M
. fijiensis
effectors. Clear differences in allelic diversity were observed between strains originating from
S
outh‐
E
ast
A
sia relative to strains from other banana‐producing continents, consistent with the hypothesis that
M
. fijiensis
originated in the
A
sian‐
P
acific region. Furthermore, transient co‐expression of the
MfA
vr4
effector alleles and the tomato
C
f‐4
resistance gene, as well as of
MfE
cp2
,
MfE
cp2‐2
and
MfE
cp2‐3
and the putative
C
f‐
E
cp2
resistance gene, indicated that effector alleles able to overcome these resistance genes are already present in natural populations of the pathogen, thus questioning the durability of resistance that can be provided by these genes in the field.
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BFBNIB, DOBA, FZAB, GIS, IJS, IZUM, KILJ, NLZOH, NUK, OILJ, PILJ, PNG, SAZU, SBCE, SBMB, UILJ, UKNU, UL, UM, UPUK
Summary
α‐Tomatine is an antifungal glycoalkaloid that provides basal defense to tomato (
S
olanum lycopersicum
). However, tomato pathogens overcome this basal defense barrier by the secretion of ...tomatinases that degrade α‐tomatine into the less fungitoxic compounds β‐tomatine and tomatidine. Although pathogenic on tomato, it has been reported that the biotrophic fungus
C
ladosporium fulvum
is unable to detoxify α‐tomatine.
Here, we present a functional analysis of the glycosyl hydrolase (
GH
10),
C
f
T
om1, which is orthologous to fungal tomatinases.
We show that
C
. fulvum
hydrolyzes α‐tomatine into tomatidine
in vitro
and during the infection of tomato, which is fully attributed to the activity of
C
f
T
om1, as shown by the heterologous expression of this enzyme in tomato. Accordingly, ∆
cftom1
mutants of
C
. fulvum
are more sensitive to α‐tomatine and are less virulent than the wild‐type fungus on tomato. Although α‐tomatine is thought to be localized in the vacuole, we show that it is also present in the apoplast, where it is hydrolyzed by
C
f
T
om1 on infection. The accumulation of tomatidine during infection appears to be toxic to tomato cells and does not suppress defense responses, as suggested previously.
Altogether, our results show that
C
f
T
om1 is responsible for the detoxification of α‐tomatine by
C
. fulvum
, and is required for full virulence of this fungus on tomato.
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BFBNIB, FZAB, GIS, IJS, KILJ, NLZOH, NMLJ, NUK, OILJ, PNG, SAZU, SBCE, SBMB, UL, UM, UPUK
In order to establish disease, plant pathogenic fungi deliver effectors in the apoplastic space surrounding host cells as well as into host cells themselves to manipulate host physiology in favour of ...their own growth. Cladosporium fulvum is a non-obligate biotrophic fungus causing leaf mould disease of tomato. For decades, this fungus has been a model to study the molecular basis of plant-pathogen interactions involving effector proteins. Characterization of these effectors revealed their roles in both virulence and avirulence as they facilitate colonization of the host in the absence of cognate tomato Cf resistance genes, but also trigger Cf-mediated resistance in the presence of these genes. The availability of the genome sequence of C. fulvum is a great resource allowing us to dissect and better understand the molecular interaction between this fungus and tomato, particularly with regards to identification of new effectors. Such knowledge is of important to improve current strategies not only for disease resistance breeding of tomato against C. fulvum, but also for other host plants that are attacked by pathogenic fungi with similar infection strategies and lifestyles. In chapter 1 we give an introduction to the C. fulvum-tomato pathosystem. In a compatible interaction, C. fulvum secretes small cysteine-rich effectors that positively contribute to fungal virulence. Two of these effectors are chitin-binding proteins including Avr4, which protects fungal cell walls against hydrolysis by plant chitinases, and Ecp6, which sequesters released small chitin fragments, thereby preventing induction of basal defense responses associated with their recognition by plant receptors. Another effector, Avr2, is an inhibitor of four tomato cysteine proteases that are also important for basal plant defense. However, in an incompatible interaction, these effectors are directly or indirectly perceived by corresponding resistance proteins (encoded by Cf resistance genes that belong to the class of receptor-like proteins; RLPs) mediating race-specific plant defense responses also known as effector-triggered immunity. In chapter 2 we exploit the availability of the genome sequence of C. fulvum to identify novel effectors involved in virulence and avirulence of this fungus. An in silico search was performed using common features of characterized C. fulvum effectors: they (i) contain a signal peptide, (ii) are small (<300 amino acids) and (iii) contain at least four cysteine residues (SSCPs). This search identified 271 SSCPs in the C. fulvum genome. A subset of 60 of these predicted effectors was heterologously expressed in tomato lines carrying different R-traits, including Cf-1, Cf-3, Cf-5, Cf-9B, Cf-11 and Cf-Ecp3 in order to identify the corresponding effectors that are recognized by the RLPs. Although the screen of this subset of SSCPs did not result in identification of a new avirulence gene, two non-specific necrosis-inducing proteins were identified. In addition, a homology search identified CfNLP1, a gene encoding a functional NEP1-like protein that triggers non-specific necrosis in plants. However, quantitative PCR showed that these three genes are lowly or not expressed during tomato infection, which was also true for the in planta expression of some of the effector candidates that were tested for recognition by Cf proteins. In contrast, all genes from C. fulvum encoding the effectors that have been reported so far are highly up-regulated during infection where they play an important role in establishing disease. Like Avr2, Avr4, Ecp2 and Ecp6, we report that Ecp4 and Ecp5 also are involved in virulence of C. fulvum on tomato. Finally, we discuss the limitations of only using bioinformatics approaches to identify novel effectors involved in virulence. Inchapter 3 we describe the identification and characterization of a novel effector secreted by C. fulvum. CfTom1 encodes a functional tomatinase enzyme, which belongs to family 10 of glycoside hydrolases (GH10). Bacterial and fungal pathogens of tomato secrete this enzyme to detoxify the toxic saponin, α-tomatine, into the less toxic compounds tomatidine and lycotetraose. Similarly, CfTom1 is responsible for α-tomatine deoxification by C. fulvum both in vitro and during infection of tomato. Accordingly, ∆cftom1 mutants are more sensitive to α-tomatine because they can no longer detoxify α-tomatine. They are less virulent on tomato plants than wild-type as reflected by a delay in disease symptom development and reduced fungal biomass production. In addition, tomatidine appears to be more toxic to tomato cells than α-tomatine, but it does not suppress plant defense responses as previously suggested in literature. Altogether, our results clearly indicate that CfTom1, the major or possibly only tomatinase enzyme produced by C. fulvum, contributes to full virulence of this fungus on tomato by detoxifying α-tomatine. Hardly anything is known about in planta regulation of effector genes. In chapter 4 we describe the functional characterization of CfWor1, a homologue of FoSge1, a conserved transcriptional regulator of effectors in Fusarium oxysporum f. sp. lycopersici. CfWor1 is also homologous to Wor1/Ryp1/Mit1 proteins, which are involved in morphological switches in Candida albicans, Histoplasma capsulatum and Saccharomyces cerevisiae, respectively. In contrast to FoSge1, CfWor1 is unlikely a positive regulator of effector genes because it is weakly expressed during infection of tomato. Compared to wild-type, ∆cfwor1 mutants show strong developmental and morphological defects. ∆cfwor1 mutants do not produce any conidia, but differentiate sclerotium-like structures and secrete an extracellular matrix that covers fungal hyphae.∆cfwor1 mutants are no longer virulent on tomato, likely because of developmental defects. Although constitutive expression of CfWor1 in C. fulvum did not cause any obvious developmental defects, except reduced conidia production, the transformants showed reduced virulence. Quantitative PCR on known effector and secondary metabolism genes in both ∆cfwor1 mutants and constitutive expression transformant revealed that the effect of CfWor1 on the expression of these genes is likely due to developmental defects rather than direct regulation. Complementationof a non-virulent ∆fosge1 mutant of F. oxysporum f. sp. lycopersici with full length CfWor1 or chimera of CfWor1 and FoSge1 restored expression of SIX effector genes, but not virulence, indicating that reduced virulence observed for the ∆fosge1 mutant is not solely due to loss of expression ofthese effector genes. Altogether, our study suggests that CfWor1 is a major regulator of development in C. fulvum which indirectly affects virulence. Chapter 5 provides a general discussion of the present work on C. fulvum effectors, with particular emphasis on comparative genomics and transcriptomics approaches to identify novel effectors involved in fungal virulence and avirulence. Our findings are put in a broader perspective including a discussion on how identification of effectors will improve our understanding of molecular interactions between plants and pathogenic fungi and how we can use this knowledge to develop new strategies for sustainable disease resistance breeding.
Summary alpha-Tomatine is an antifungal glycoalkaloid that provides basal defense to tomato (Solanum lycopersicum). However, tomato pathogens overcome this basal defense barrier by the secretion of ...tomatinases that degrade alpha-tomatine into the less fungitoxic compounds beta-tomatine and tomatidine. Although pathogenic on tomato, it has been reported that the biotrophic fungus Cladosporium fulvum is unable to detoxify alpha-tomatine. Here, we present a functional analysis of the glycosyl hydrolase (GH10), CfTom1, which is orthologous to fungal tomatinases. We show that C. fulvum hydrolyzes alpha-tomatine into tomatidine in vitro and during the infection of tomato, which is fully attributed to the activity of CfTom1, as shown by the heterologous expression of this enzyme in tomato. Accordingly, incrementcftom1 mutants of C. fulvum are more sensitive to alpha-tomatine and are less virulent than the wild-type fungus on tomato. Although alpha-tomatine is thought to be localized in the vacuole, we show that it is also present in the apoplast, where it is hydrolyzed by CfTom1 on infection. The accumulation of tomatidine during infection appears to be toxic to tomato cells and does not suppress defense responses, as suggested previously. Altogether, our results show that CfTom1 is responsible for the detoxification of alpha-tomatine by C. fulvum, and is required for full virulence of this fungus on tomato. PUBLICATION ABSTRACT
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BFBNIB, FZAB, GIS, IJS, KILJ, NLZOH, NMLJ, NUK, OILJ, PNG, SAZU, SBCE, SBMB, UL, UM, UPUK
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