Virulence characteristics of Blumeria graminis f. sp. tritici and its genetic diversity by EST-SSR analyses.

Wheat powdery mildew, caused by Blumeria graminis f. sp. tritici (an obligate biotrophic pathogen) is a worldwide threat to wheat production that occurs over a wide geographic area in China. For monitoring genetic variation and virulence structure of Blumeria graminis f. sp. tritici in Liaoning, Heilongjiang, and Sichuan in 2015, 31 wheat lines with known Powdery mildew resistance genes and 2 EST-SSR markers were used to characterize the virulence and genetic diversity. Results indicated that 90% of all isolates were virulent on Pm3c, Pm3e, Pm3f, Pm4a, Pm5, Pm6 (Timgalen), Pm7, Pm16, Pm19, and Pm1 + 2 + 9 and 62.6% to 89.9% of isolates were virulent on Pm3a, Pm3b, Pm3d, Pm4b, Pm6 (Coker747), Pm8, Pm17, Pm20, Pm23, Pm30, Pm4 + 8, Pm5 + 6, Pm4b + mli, Pm2 + mld, Pm4 + 2X, Pm2 + 6. The Pm13 and PmXBD genes were effective against most collected isolates from Liaoning and Heilongjiang Provinces. Only Pm21 exhibited an immune infection response to all isolates. Furthermore, closely related isolates within each region were distinguished by cluster analyses using EST-SSR representing some gene exchanges and genetic relationships between the flora in Northeast China (Liaoning, Heilongjiang) and Sichuan. Only 45% of the isolates tested show a clear correlation between EST-SSR genetic polymorphisms and the frequency of virulence gene data. However, the EST-SSR polymorphism of isolated genes did not correspond to the virulence diversity of isolates in the single-gene lineage identification of hosts.

An Integrated Omics Approach Uncovers the Novel Effector Ecp20-2 Required for Full Virulence of Cladosporium fulvum on Tomato.

The fungus Cladosporium fulvum causes the leaf mould in tomatoes. During the colonization of the host, it secretes plenty of effector proteins into the plant apoplast to suppress the plant's immune system. Here, we characterized and functionally analyzed the Ecp20-2 gene of C. fulvum using combined omics approaches. RNA-sequencing of susceptible tomato plants inoculated with C. fulvum race 0WU showed strongly induced expression of the Ecp20-2 gene. Strong upregulation of expression of the Ecp20-2 gene was confirmed by qPCR, and levels were comparable to those of other known effectors of C. fulvum. The Ecp20-2 gene encodes a small secreted protein of 149 amino acids with a predicted signal peptide of 17 amino acids. Mass spectrometry of apoplastic fluids from infected tomato leaves revealed the presence of several peptides originating from the Ecp20-2 protein, indicating that the protein is secreted and likely functions in the apoplast. In the genome of C. fulvum, Ecp20-2 is surrounded by various repetitive elements, but no allelic variation was detected in the coding region of Ecp20-2 among 120 C. fulvum isolates collected in Japan. Δecp20-2 deletion mutants of strain 0WU of C. fulvum showed decreased virulence, supporting that Ecp20-2 is an effector required for full virulence of the fungus. Virulence assays confirmed a significant reduction of fungal biomass in plants inoculated with Δecp20-2 mutants compared to those inoculated with wild-type, Δecp20-2-complemented mutants, and ectopic transformants. Sequence similarity analysis showed the presence of Ecp20-2 homologs in the genomes of several Dothideomycete fungi. The Ecp20-2 protein shows the best 3D homology with the PevD1 effector of Verticillium dahliae, which interacts with and inhibits the activity of the pathogenesis-related protein PR5, which is involved in the immunity of several host plants.

NADP-MALIC ENZYME 1 Affects Germination after Seed Storage in Arabidopsis thaliana.

Aging decreases the quality of seeds and results in agricultural and economic losses. The damage that occurs at the biochemical level can alter the seed physiological status. Although loss of viability has been investigated frequently, little information exists on the molecular and biochemical factors involved in seed deterioration and loss of viability. Oxidative stress has been implicated as a major contributor to seed deterioration, and several pathways are involved in protection against this. In this study, we show that seeds of Arabidopsis thaliana lacking a functional NADP-MALIC ENZYME 1 (NADP-ME1) have reduced seed viability relative to the wild type. Seeds of the NADP-ME1 loss-of-function mutant display higher levels of protein carbonylation than those of the wild type. NADP-ME1 catalyzes the oxidative decarboxylation of malate to pyruvate with the simultaneous production of CO2 and NADPH. Upon seed imbibition, malate and amino acids accumulate in embryos of aged seeds of the NADP-ME1 loss-of-function mutant compared with those of the wild type. NADP-ME1 expression is increased in imbibed aged as compared with non-aged seeds. NADP-ME1 activity at testa rupture promotes normal germination of aged seeds. In seedlings of aged seeds, NADP-ME1 is specifically active in the root meristematic zone. We propose that NADP-ME1 activity is required for protecting seeds against oxidation during seed dry storage.

Specific Hypersensitive Response-Associated Recognition of New Apoplastic Effectors from Cladosporium fulvum in Wild Tomato.

Tomato leaf mold disease is caused by the biotrophic fungus Cladosporium fulvum. During infection, C. fulvum produces extracellular small secreted protein (SSP) effectors that function to promote colonization of the leaf apoplast. Resistance to the disease is governed by Cf immune receptor genes that encode receptor-like proteins (RLPs). These RLPs recognize specific SSP effectors to initiate a hypersensitive response (HR) that renders the pathogen avirulent. C. fulvum strains capable of overcoming one or more of all cloned Cf genes have now emerged. To combat these strains, new Cf genes are required. An effectoromics approach was employed to identify wild tomato accessions carrying new Cf genes. Proteomics and transcriptome sequencing were first used to identify 70 apoplastic in planta-induced C. fulvum SSPs. Based on sequence homology, 61 of these SSPs were novel or lacked known functional domains. Seven, however, had predicted structural homology to antimicrobial proteins, suggesting a possible role in mediating antagonistic microbe-microbe interactions in planta. Wild tomato accessions were then screened for HR-associated recognition of 41 SSPs, using the Potato virus X-based transient expression system. Nine SSPs were recognized by one or more accessions, suggesting that these plants carry new Cf genes available for incorporation into cultivated tomato.

A conserved proline residue in Dothideomycete Avr4 effector proteins is required to trigger a Cf-4-dependent hypersensitive response.

CfAvr4, a chitin-binding effector protein produced by the Dothideomycete tomato pathogen Cladosporium fulvum, protects the cell wall of this fungus against hydrolysis by secreted host chitinases during infection. However, in the presence of the Cf-4 immune receptor of tomato, CfAvr4 triggers a hypersensitive response (HR), which renders the pathogen avirulent. Recently, several orthologues of CfAvr4 have been identified from phylogenetically closely related species of Dothideomycete fungi. Of these, DsAvr4 from Dothistroma septosporum also triggers a Cf-4-dependent HR, but CaAvr4 and CbAvr4 from Cercospora apii and Cercospora beticola, respectively, do not. All, however, bind chitin. To identify the region(s) and specific amino acid residue(s) of CfAvr4 and DsAvr4 required to trigger a Cf-4-dependent HR, chimeric and mutant proteins, in which specific protein regions or single amino acid residues, respectively, were exchanged between CfAvr4 and CaAvr4 or DsAvr4 and CbAvr4, were tested for their ability to trigger an HR in Nicotiana benthamiana plants transgenic for the Cf-4 immune receptor gene. Based on this approach, a single region common to CfAvr4 and DsAvr4 was determined to carry a conserved proline residue necessary for the elicitation of this HR. In support of this result, a Cf-4-dependent HR was triggered by mutant CaAvr4 and CbAvr4 proteins carrying an arginine-to-proline substitution at this position. This study provides the first step in deciphering how Avr4 orthologues from different Dothideomycete fungi trigger a Cf-4-dependent HR.

Pseudogenization in pathogenic fungi with different host plants and lifestyles might reflect their evolutionary past.

Pseudogenes are genes with significant homology to functional genes, but contain disruptive mutations (DMs) leading to the production of non- or partially functional proteins. Little is known about pseudogenization in pathogenic fungi with different lifestyles. Here, we report the identification of DMs causing pseudogenes in the genomes of the fungal plant pathogens Botrytis cinerea, Cladosporium fulvum, Dothistroma septosporum, Mycosphaerella fijiensis, Verticillium dahliae and Zymoseptoria tritici. In these fungi, we identified 1740 gene models containing 2795 DMs obtained by an alignment-based gene prediction method. The contribution of sequencing errors to DMs was minimized by analyses of resequenced genomes to obtain a refined dataset of 924 gene models containing 1666 true DMs. The frequency of pseudogenes varied from 1% to 5% in the gene catalogues of these fungi, being the highest in the asexually reproducing fungus C. fulvum (4.9%), followed by D. septosporum (2.4%) and V. dahliae (2.1%). The majority of pseudogenes do not represent recent gene duplications, but members of multi-gene families and unitary genes. In general, there was no bias for pseudogenization of specific genes in the six fungi. Single exceptions were those encoding secreted proteins, including proteases, which appeared more frequently pseudogenized in C. fulvum than in D. septosporum. Most pseudogenes present in these two phylogenetically closely related fungi are not shared, suggesting that they are related to adaptation to a different host (tomato versus pine) and lifestyle (biotroph versus hemibiotroph).

Secondary metabolism and biotrophic lifestyle in the tomato pathogen Cladosporium fulvum.

Cladosporium fulvum is a biotrophic fungal pathogen that causes leaf mould of tomato. Analysis of its genome suggested a high potential for production of secondary metabolites (SM), which might be harmful to plants and animals. Here, we have analysed in detail the predicted SM gene clusters of C. fulvum employing phylogenetic and comparative genomic approaches. Expression of the SM core genes was measured by RT-qrtPCR and produced SMs were determined by LC-MS and NMR analyses. The genome of C. fulvum contains six gene clusters that are conserved in other fungal species, which have undergone rearrangements and gene losses associated with the presence of transposable elements. Although being a biotroph, C. fulvum has the potential to produce elsinochrome and cercosporin toxins. However, the corresponding core genes are not expressed during infection of tomato. Only two core genes, PKS6 and NPS9, show high expression in planta, but both are significantly down regulated during colonization of the mesophyll tissue. In vitro SM profiling detected only one major compound that was identified as cladofulvin. PKS6 is likely involved in the production of this pigment because it is the only core gene significantly expressed under these conditions. Cladofulvin does not cause necrosis on Solanaceae plants and does not show any antimicrobial activity. In contrast to other biotrophic fungi that have a reduced SM production capacity, our studies on C. fulvum suggest that down-regulation of SM biosynthetic pathways might represent another mechanism associated with a biotrophic lifestyle.