G.H.J. Kema

Tomato Cf resistance proteins mediate recognition of cognate homologous effectors from fungi pathogenic on dicots and monocots.

Most fungal effectors characterized so far are species-specific and facilitate virulence on a particular host plant. During infection of its host tomato, Cladosporium fulvum secretes effectors that function as virulence factors in the absence of cognate Cf resistance proteins and induce effector-triggered immunity in their presence. Here we show that homologs of the C. fulvum Avr4 and Ecp2 effectors are present in other pathogenic fungi of the Dothideomycete class, including Mycosphaerella fijiensis, the causal agent of black Sigatoka disease of banana. We demonstrate that the Avr4 homolog of M. fijiensis is a functional ortholog of C. fulvum Avr4 that protects fungal cell walls against hydrolysis by plant chitinases through binding to chitin and, despite the low overall sequence homology, triggers a Cf-4-mediated hypersensitive response (HR) in tomato. Furthermore, three homologs of C. fulvum Ecp2 are found in M. fijiensis, one of which induces different levels of necrosis or HR in tomato lines that lack or contain a putative cognate Cf-Ecp2 protein, respectively. In contrast to Avr4, which acts as a defensive virulence factor, M. fijiensis Ecp2 likely promotes virulence by interacting with a putative host target causing host cell necrosis, whereas Cf-Ecp2 could possibly guard the virulence target of Ecp2 and trigger a Cf-Ecp2-mediated HR. Overall our data suggest that Avr4 and Ecp2 represent core effectors that are collectively recognized by single cognate Cf-proteins. Transfer of these Cf genes to plant species that are attacked by fungi containing these cognate core effectors provides unique ways for breeding disease-resistant crops.

Zymoseptoria gen. nov.: a new genus to accommodate Septoria-like species occurring on graminicolous hosts

The Mycosphaerella complex is both poly- and paraphyletic, containing several different families and genera. The genus Mycosphaerella is restricted to species with Ramularia anamorphs, while Septoria is restricted to taxa that cluster with the type species of Septoria, S. cytisi, being closely related to Cercospora in the Mycosphaerellaceae. Species that occur on graminicolous hosts represent an as yet undescribed genus, for which the name Zymoseptoria is proposed. Based on the 28S nrDNA phylogeny derived in this study, Zymoseptoria is shown to cluster apart from Septoria. Morphologically species of Zymoseptoria can also be distinguished by their yeast-like growth in culture, and the formation of different conidial types that are absent in Septoria s.str. Other than the well-known pathogens such as Z. tritici, the causal agent of septoria tritici blotch on wheat, and Z. passerinii, the causal agent of septoria speckled leaf blotch of barley, both for which epitypes are designated, two leaf blotch pathogens are also described on graminicolous hosts from Iran. Zymoseptoria brevis sp. nov. is described from Phalaris minor, and Z. halophila comb. nov. from leaves of Hordeum glaucum. Further collections are now required to elucidate the relative importance, host range and distribution of these species.

Gα and Gβ Proteins Regulate the Cyclic AMP Pathway That Is Required for Development and Pathogenicity of the Phytopathogen Mycosphaerella graminicola

ABSTRACT We identified and functionally characterized genes encoding three Gα proteins and one Gβ protein in the dimorphic fungal wheat pathogen Mycosphaerella graminicola, which we designated MgGpa1, MgGpa2, MgGpa3, and MgGpb1, respectively. Sequence comparisons and phylogenetic analyses showed that MgGPA1 and MgGPA3 are most related to the mammalian Gαi and Gαs families, respectively, whereas MgGPA2 is not related to either of these families. On potato dextrose agar (PDA) and in yeast glucose broth (YGB), MgGpa1 mutants produced significantly longer spores than those of the wild type (WT), and these developed into unique fluffy mycelia in the latter medium, indicating that this gene negatively controls filamentation. MgGpa3 mutants showed more pronounced yeast-like growth accompanied with hampered filamentation and secreted a dark-brown pigment into YGB. Germ tubes emerging from spores of MgGpb1 mutants were wavy on water agar and showed a nested type of growth on PDA that was due to hampered filamentation, numerous cell fusions, and increased anastomosis. Intracellular cyclic AMP (cAMP) levels of MgGpb1 and MgGpa3 mutants were decreased, indicating that both genes positively regulate the cAMP pathway, which was confirmed because the WT phenotype was restored by adding cAMP to these mutant cultures. The cAMP levels in MgGpa1 mutants and the WT were not significantly different, suggesting that this gene might be dispensable for cAMP regulation. In planta assays showed that mutants of MgGpa1, MgGpa3, and MgGpb1 are strongly reduced in pathogenicity. We concluded that the heterotrimeric G proteins encoded by MgGpa3 and MgGpb1 regulate the cAMP pathway that is required for development and pathogenicity in M. graminicola.

The potential of high-resolution BAC-FISH in banana breeding

The genetic complexity in the genus Musa has been subject of study in many breeding programs worldwide. Parthenocarpy, female sterility, polyploidy in different cultivars and limited amount of genetic and genomic information make the production of new banana cultivars difficult and time consuming. In addition, it is known that part of the cultivars and related wild species in the genus contain numerous chromosomal rearrangements. In order to produce new cultivars more effectively breeders must better understand the genetic differences of the potential crossing parents for introgression hybridization, but extensive genetic information is lacking. As an alternative to achieve information on genetic collinearity we make use of modern chromosome map technology known as high-resolution fluorescent in situ hybridization (FISH). This article presents the technical aspects and applications of such a technology in Musa species. The technique deals with BAC clone positioning on pachytene chromosomes of Calcutta 4 (Musa acuminata ssp. burmanicoides, A genome group, section Eumusa) and M. velutina (section Rodochlamys). Pollen mother cells digestion with pectolytic enzymes and maceration with acetic acid were optimized for making cell spread preparations appropriate for FISH. As an example of this approach we chose BAC clones that contain markers to known resistance genes and hybridize them for establishing their relative positions on the two species. Technical challenges for adapting existing protocols to the banana cells are presented. We also discuss how this technique can be instrumental for validating collinearity between potential crossing parents and how the method can be helpful in future mapping initiatives, and how this method allows identification of chromosomal rearrangements between related Musa species and cultivars.

FPLC and liquid-chromatography mass spectrometry identify candidate necrosis-inducing proteins from culture filtrates of the fungal wheat pathogen Zymoseptoria tritici

Culture filtrates (CFs) of the fungal wheat pathogen Zymoseptoria tritici were assayed for necrosis-inducing activity after infiltration in leaves of various wheat cultivars. Active fractions were partially purified and characterized. The necrosis-inducing factors in CFs are proteinaceous, heat stable and their necrosis-inducing activity is temperature and light dependent. The in planta activity of CFs was tested by a time series of proteinase K (PK) co-infiltrations, which was unable to affect activity 30min after CF infiltrations. This suggests that the necrosis inducing proteins (NIPs) are either absent from the apoplast and likely actively transported into mesophyll cells or protected from the protease by association with a receptor. Alternatively, plant cell death signaling pathways might be fully engaged during the first 30min and cannot be reversed even after PK treatment. Further fractionation of the CFs with the highest necrosis-inducing activity involved fast performance liquid chromatography, SDS-PAGE and mass spectrometry. This revealed that most of the proteins present in the fractions have not been described before. The two most prominent ZtNIP encoding candidates were heterologously expressed in Pichia pastoris and subsequent infiltration assays showed their differential activity in a range of wheat cultivars.

Stress and sexual reproduction affect the dynamics of the wheat pathogen effector AvrStb6 and strobilurin resistance

Host resistance and fungicide treatments are cornerstones of plant-disease control. Here, we show that these treatments allow sex and modulate parenthood in the fungal wheat pathogen Zymoseptoria tritici. We demonstrate that the Z. tritici–wheat interaction complies with the gene-for-gene model by identifying the effector AvrStb6, which is recognized by the wheat resistance protein Stb6. Recognition triggers host resistance, thus implying removal of avirulent strains from pathogen populations. However, Z. tritici crosses on wheat show that sex occurs even with an avirulent parent, and avirulence alleles are thereby retained in subsequent populations. Crossing fungicide-sensitive and fungicide-resistant isolates under fungicide pressure results in a rapid increase in resistance-allele frequency. Isolates under selection always act as male donors, and thus disease control modulates parenthood. Modeling these observations for agricultural and natural environments reveals extended durability of host resistance and rapid emergence of fungicide resistance. Therefore, fungal sex has major implications for disease control.Identification of AvrStb6, the fungal avirulence effector that triggers Stb6-mediated resistance in wheat, here demonstrates that neither host resistance nor fungicide treatment suppresses fungal sexual reproduction, thus unveiling implications of fungal sex in plant disease control.

Proteome catalog of Zymoseptoria tritici captured during pathogenesis in wheat

Zymoseptoria tritici is an economically important pathogen of wheat. However, the molecular basis of pathogenicity on wheat is still poorly understood. Here, we present a global survey of the proteins secreted by this fungus in the apoplast of resistant (cv. Shafir) and susceptible (cv. Obelisk) wheat cultivars after inoculation with reference Z. tritici strain IPO323. The fungal proteins present in apoplastic fluids were analyzed by gel electrophoresis and by data-independent acquisition liquid chromatography/mass spectrometry (LC/MS(E)) combined with data-dependent acquisition LC-MS/MS. Subsequent mapping mass spectrometry-derived peptide sequence data against the genome sequence of strain IPO323 identified 665 peptides in the MS(E) and 93 in the LC-MS/MS mode that matched to 85 proteins. The identified fungal proteins, including cell-wall degrading enzymes and proteases, might function in pathogenicity, but the functions of many remain unknown. Most fungal proteins accumulated in cv. Obelisk at the onset of necrotrophy. This inventory provides an excellent basis for future detailed studies on the role of these genes and their encoded proteins during pathogenesis in wheat.

A new mechanism for reduced sensitivity to demethylation-inhibitor fungicides in the fungal banana black Sigatoka pathogen Pseudocercospora fijiensis

Summary The Dothideomycete Pseudocercospora fijiensis, previously Mycosphaerella fijiensis, is the causal agent of black Sigatoka, one of the most destructive diseases of bananas and plantains. Disease management depends on fungicide applications, with a major contribution from sterol demethylation‐inhibitors (DMIs). The continued use of DMIs places considerable selection pressure on natural P. fijiensis populations, enabling the selection of novel genotypes with reduced sensitivity. The hitherto explanatory mechanism for this reduced sensitivity was the presence of non‐synonymous point mutations in the target gene Pfcyp51, encoding the sterol 14α‐demethylase enzyme. Here, we demonstrate a second mechanism involved in DMI sensitivity of P. fijiensis. We identified a 19‐bp element in the wild‐type (wt) Pfcyp51 promoter that concatenates in strains with reduced DMI sensitivity. A polymerase chain reaction (PCR) assay identified up to six Pfcyp51 promoter repeats in four field populations of P. fijiensis in Costa Rica. We used transformation experiments to swap the wt promoter of a sensitive field isolate with a promoter from a strain with reduced DMI sensitivity that comprised multiple insertions. Comparative in vivo phenotyping showed a functional and proportional up‐regulation of Pfcyp51, which consequently decreased DMI sensitivity. Our data demonstrate that point mutations in the Pfcyp51 coding domain, as well as promoter inserts, contribute to the reduced DMI sensitivity of P. fijiensis. These results provide new insights into the importance of the appropriate use of DMIs and the need for the discovery of new molecules for black Sigatoka management.