Involvement of the Autophagy Protein Atg6 in Development and Virulence in the Gray Mold Fungus Botrytis cinerea.

Gray mold caused by Botrytis cinerea is a devastating disease that leads to huge economic losses worldwide. Autophagy is an evolutionarily conserved process that maintains intracellular homeostasis through self-eating. In this study, we identified and characterized the biological function of the autophagy-related protein Atg6 in B. cinerea. Targeted deletion of the BcATG6 gene showed block of autophagy and several phenotypic defects in aspects of mycelial growth, conidiation, sclerotial formation and virulence. All of the phenotypic defects were restored by targeted gene complementation. Taken together, these results suggest that BcAtg6 plays important roles in the regulation of various cellular processes in B. cinerea.

Evolutionary and genomic comparisons of hybrid uninucleate and nonhybrid Rhizoctonia fungi.

The basidiomycetous fungal genus, Rhizoctonia, can cause severe damage to many plants and is composed of multinucleate, binucleate, and uninucleate species differing in pathogenicity. Here we generated chromosome-scale genome assemblies of the three nuclear types of Rhizoctonia isolates. The genomic comparisons revealed that the uninucleate JN strain likely arose by somatic hybridization of two binucleate isolates, and maintained a diploid nucleus. Homeolog gene pairs in the JN genome have experienced both decelerated or accelerated evolution. Homeolog expression dominance occurred between JN subgenomes, in which differentially expressed genes show potentially less evolutionary constraint than the genes without. Analysis of mating-type genes suggested that Rhizoctonia maintains the ancestral tetrapolarity of the Basidiomycota. Long terminal repeat-retrotransposons displayed a reciprocal correlation with the chromosomal GC content in the three chromosome-scale genomes. The more aggressive multinucleate XN strain had more genes encoding enzymes for host cell wall decomposition. These findings demonstrate some evolutionary changes of a recently derived hybrid and in multiple nuclear types of Rhizoctonia.

Comparison of mitochondrial genomes from multi-, Bi-, and uninucleate Rhizoctonia.

Six circular mitochondrial genomes of multi-, bi-, and uninucleate Rhizoctonia isolates were assembled and found that all the genomes contain 14 conserved protein-coding genes, one ribosomal protein (rps3), and 23 tRNA in the same order. The mitogenome sizes of uninucleate isolates were relatively smaller than binucleate and multinucleate stains. The size variations between uninucleate and multinucleate isolates were from both intergenic and intronic regions, whereas the differences between uninucleate and binucleate isolates were predominantly from intergenic regions. The phylogenetic analysis revealed that Rhizoctonia strains of the same nucleate types had a closer relationship.

Effects of Temperature and Moisture on Conidia Germination, Infection, and Acervulus Formation of the Apple Marssonina Leaf Blotch Pathogen (Diplocarpon mali) in China.

Apple Marssonina leaf blotch (AMLB; Diplocarpon mali) is a severe disease of apple that mainly causes premature leaf defoliation in many apple growing areas worldwide. AMLB epidemic development is closely related to temperature and rainfall. In this study, the effects of temperature and moisture on conidium germination, infection on leaves, and acervulus production were investigated under controlled environments. The temperature required for conidium germination and infection ranged from 5 to 30°C, with the optimum at approximately 23°C. The temperature required for acervulus formation was slightly higher, with the optimum at 24.6°C. Wetness was needed in order for conidia to germinate and infect; only a few conidia germinated at 100% RH. However, lesions can produce acervuli in dry conditions. The minimum duration of leaf wetness required for conidia to complete the entire infection process was 14, 8, 4, and 6 h at 10, 15, 20, and 25°C, respectively. A model describing the effect of temperature and leaf wetness duration was built. The model estimated that the optimum temperature for conidial infection was 22.6°C and the minimum wetness duration required was 4.8 h. This model can be used to forecast D. mali conidial infection to assist in disease management in commercial apple production.

Genome Sequence Resource of Coniella vitis, a Fungal Pathogen Causing Grape White Rot Disease.

Several fungal pathogens cause grape white rot disease and Coniella vitis is a predominant pathogen in Chinese vineyards. The disease occurs on leaves, vines, and fruit berries, leading to considerable yield losses and even to total destruction of vineyards. Here, we present the first Pacbio and Illumina-sequenced draft genome assembly of C. vitis QNYT13637 and its annotation. This genome sequence provides a unique resource that will be a powerful foundation for future research on exploring virulence-related genes, investigating the pathogenicity mechanism of the pathogen, and, finally, improvement of white rot disease management strategies.

FgEaf6 regulates virulence, asexual/sexual development and conidial septation in Fusarium graminearum.

Fusarium graminearum is a destructive fungal pathogen and a major cause of Fusarium head blight (FHB) which results in severe grain yield losses and quality reduction. Additionally, the pathogen produces mycotoxins during plant infection, which are harmful to the health of humans and livestock. As it is well known that lysine acetyltransferase complexes play important roles in pathogenesis, the roles of the Eaf6 homolog-containing complex have not been reported in fungal pathogen. In this study, a Eaf6 homolog FgEaf6 was identified in F. graminearum. To investigate the functions of FgEaf6, the gene was deleted using the split-marker method. ΔFgEaf6 mutant exhibited manifold defects in hyphal growth, conidial septation, asexual and sexual reproduction. Moreover, the virulence of the ΔFgEaf6 mutant was drastically reduced in both wheat heads and wheat coleoptiles. However, the FgEaf6 gene deletion did not impact DON production. An FgEaf6-gfp fusion localized to the nucleus and a conserved coiled-coil (C-C) domain was predicted in the sequence. Mutants with deletions in the C-C domain displayed similar defects during development and virulence as observed in the ΔFgEaf6 mutant. Moreover, the truncated gene was cytoplasm localized. In conclusion, the FgEaf6 encodes a nuclear protein, which plays key regulatory roles in hyphal growth, conidial septation, asexual/sexual reproduction, and the virulence of F. graminearum. The C-C is an indispensable domain in the gene. This is the first report on Eaf6 homolog functioning in virulence of fungal pathogen.

Comparative acetylome analysis reveals the potential roles of lysine acetylation for DON biosynthesis in Fusarium graminearum.

BACKGROUND: Fusarium graminearum is a destructive fungal pathogen of wheat, barley and other small grain cereals. During plant infection, the pathogen produces trichothecene mycotoxin deoxynivalenol (DON), which is harmful to human and livestock. FgGCN5 encodes a GCN5 acetyltransferase. The gene deletion mutant Fggcn5 failed to produce DON. We assumed that lysine acetylation might play a key regulatory role in DON biosynthesis in the fungus. RESULTS: In this study, the acetylome comparison between Fggcn5 mutant and wild-type strain PH-1 was performed by using affinity enrichment and high resolution LC-MS/MS analysis. Totally, 1875 acetylated proteins were identified in Fggcn5 mutant and PH-1. Among them, 224 and 267 acetylated proteins were identified exclusively in Fggcn5 mutant and PH-1, respectively. Moreover, 95 differentially acetylated proteins were detected at a significantly different level in the gene deletion mutant:43 were up-regulated and 52 were down-regulated. GO enrichment and KEGG-pathways enrichment analyses revealed that acetylation plays a key role in metabolism process in F. graminearum. CONCLUSIONS: Seeing that the gens playing critical roles in DON biosynthesis either in Fggcn5 mutant or PH-1. Therefore, we can draw the conclusion that the regulatory roles of lysine acetylation in DON biosynthesis in F. graminearum results from the positive and negative regulation of the related genes. The study would be a foundation to insight into the regulatory mechanism of lysine acetylation on DON biosynthesis.

Inhibitory effect and possible mechanism of a Pseudomonas strain QBA5 against gray mold on tomato leaves and fruits caused by Botrytis cinerea.

The fungal pathogen Botrytis cinerea causes gray mold disease on various hosts, which results in serious economic losses. Over the past several decades, many kinds of fungicides have been used to successfully control the disease. Meanwhile, the uses of fungicides lead to environmental pollution as well as a potential threat to the human health by the chemical residues in tomato fruit. Also, the gray mold disease is difficult to control with fungicides. Therefore, exploring alternative measures such as biological controls could be the best choice to control the disease and alleviate damages caused by fungicides. In this study, we isolated and identified a novel Pseudomonas strain termed as QBA5 from healthy tomato plant based on the morphological, biochemical characteristics and molecular detection. The antifungal activity assays revealed that, in the presence of QBA5, conidia germination, germ tube elongation and mycelial growth of B. cinerea were significantly inhibited. Most importantly, QBA5 exerted a significant preventive effectiveness against gray mold on tomato fruits and plants. The possible mechanism of QBA5 involved in the inhibition of B. cinerea was investigated. It revealed that the conidia plasma membrane of B. cinerea was severely damaged by QBA5. Further, four different antifungal compounds in the supernatant of QBA5 were separated by preparative high performance liquid chromatography (PHPLC). Overall, the data indicate that there is a considerable potential for QBA5 to reduce the damage caused by gray mold disease on tomato.

Systematic analysis of the lysine acetylome in Fusarium graminearum.

BACKGROUND: Lysine acetylation in proteins is a ubiquitous and conserved post-translational modification, playing a critical regulatory role in almost every aspect of living cells. Although known for many years, its function remains elusive in Fusarium graminearum, one of the most important necrotrophic plant pathogens with huge economic impact. RESULTS: By the combination of affinity enrichment and high-resolution LC-MS/MS analysis, large-scale lysine acetylome analysis was performed. In total, 577 lysine acetylation sites matched to 364 different proteins were identified. Bioinformatics analysis of the acetylome showed that the acetylated proteins are involved in a wide range of cellular functions and exhibit diverse subcellular localizations. Remarkably, 10 proteins involved in the virulence or DON (deoxynivalenol) biosynthesis were found to be acetylated, including 4 transcription factors, 4 protein kinases and 2 phosphatases. Protein-protein interaction network analysis revealed that acetylated protein complexes are involved in diversified interactions. CONCLUSIONS: This work provides the first comprehensive survey of a possible lysine acetylome in F. graminearum and reveals previously unappreciated roles of lysine acetylation in the regulation of diverse biological processes. This work provides a resource for functional analysis of acetylated proteins in filamentous fungi.

Molecular evolution and functional divergence of tubulin superfamily in the fungal tree of life.

Microtubules are essential for various cellular activities and ß-tubulins are the target of benzimidazole fungicides. However, the evolution and molecular mechanisms driving functional diversification in fungal tubulins are not clear. In this study, we systematically identified tubulin genes from 59 representative fungi across the fungal kingdom. Phylogenetic analysis showed that α-/ß-tubulin genes underwent multiple independent duplications and losses in different fungal lineages and formed distinct paralogous/orthologous clades. The last common ancestor of basidiomycetes and ascomycetes likely possessed two paralogs of α-tubulin (α1/α2) and ß-tubulin (ß1/ß2) genes but α2-tubulin genes were lost in basidiomycetes and ß2-tubulin genes were lost in most ascomycetes. Molecular evolutionary analysis indicated that α1, α2, and ß2-tubulins have been under strong divergent selection and adaptive positive selection. Many positively selected sites are at or adjacent to important functional sites and likely contribute to functional diversification. We further experimentally confirmed functional divergence of two ß-tubulins in Fusarium and identified type II variations in FgTub2 responsible for function shifts. In this study, we also identified δ-/ε-/η-tubulins in Chytridiomycetes. Overall, our results illustrated that different evolutionary mechanisms drive functional diversification of α-/ß-tubulin genes in different fungal lineages, and residues under positive selection could provide targets for further experimental study.

A novel wall-associated receptor-like protein kinase gene, OsWAK1, plays important roles in rice blast disease resistance.

Wall-associated protein kinases (WAKs) are a new group of receptor-like kinases (RLK) recently identified in Arabidopsis. A cDNA encoding a novel WAK was isolated from rice and was named OsWAK1 (Oryza sativa WAK). The deduced amino acid sequence of OsWAK1 showed 27.6% identity to WAK2 from Arabidopsis. OsWAK1 not only has the ability of autophosphorylation but also can phosphorylate OsRFP1, a putative transcription regulator recently identified in rice. OsRFP1 strongly interacts with the kinase domain of OsWAK1. This demonstrated that OsWAK1 is a functional protein kinase. A fusion protein of OsWAK1 with GFP was found to be localized on the cell surface. Plasmolysis experiments further revealed OsWAK1 is associated with the cell wall. Northern blotting analysis showed that infection of the rice blast fungus, Magnaporthe oryzae significantly induced the OsWAK1 transcripts, and the accumulation of OsWAK1 mRNA occurred earlier and was more abundant in rice leaves infected with an incompatible race than with a compatible race of the blast fungus. OsWAK1 was also induced after treatment by mechanical wounding, SA and MeJA, but not by ABA. These results imply that OsWAK1 is a novel gene involved in plant defense. Furthermore, six transgenic rice lines with constitutive expression of OsWAK1 became resistant to the compatible race. However, OsWAK1 expression was undetectable in leaves, stems and flowers but very weak in roots under normal growth conditions. This provides functional evidence that induction of OsWAK1 as a novel RLK plays important roles in plant disease resistance.

Molecular cloning and differential expression of an gamma-aminobutyrate transaminase gene, OsGABA-T, in rice (Oryza sativa) leaves infected with blast fungus.

gamma-Aminobutyrate transaminase (GABA-T) catalyzes the conversion of GABA to succinic semialdehyde. Using differential display PCR and cDNA library screening, a full-length GABA-T cDNA (OsGABA-T) was isolated from rice (Oryza sativa) leaves infected with an incompatible race of Magnaporthe grisea. The deduced amino acid sequence comprises 483 amino acid residues and shares 85-69% identity with GABA-T sequences from other plants. OsGABA-T expression is induced by blast fungus infection, mechanical wounding and ultraviolet radiation in rice leaves and is not detected in normal rice organs. This gene is also induced by defense signal molecules such as salicylic acid and abscisic acid, but not by jasmonic acid. Our data suggest that OsGABA-T (GABA shunt) may play a role in restricting the levels of cell death during the host-pathogen interaction.