An effector SsCVNH promotes the virulence of Sclerotinia sclerotiorum through targeting class III peroxidase AtPRX71.

Many plant pathogens secrete effector proteins into the host plant to suppress host immunity and facilitate pathogen colonization. The necrotrophic pathogen Sclerotinia sclerotiorum causes severe plant diseases and results in enormous economic losses, in which secreted proteins play a crucial role. SsCVNH was previously reported as a secreted protein, and its expression is significantly upregulated at 3 h after inoculation on the host plant. Here, we further demonstrated that deletion of SsCVNH leads to attenuated virulence. Heterologous expression of SsCVNH in Arabidopsis enhanced pathogen infection, inhibited the host PAMP-triggered immunity (PTI) response and increased plant susceptibility to S. sclerotiorum. SsCVNH interacted with class III peroxidase AtPRX71, a positive regulator of innate immunity against plant pathogens. SsCVNH could also interact with other class III peroxidases, thus reducing peroxidase activity and suppressing plant immunity. Our results reveal a new infection strategy employed by S. sclerotiorum in which the fungus suppresses the function of class III peroxidases, the major component of PTI to promote its own infection.

The Dynamic Changes of Brassica napus Seed Microbiota across the Entire Seed Life in the Field.

The seed microbiota is an important component given by nature to plants, protecting seeds from damage by other organisms and abiotic stress. However, little is known about the dynamic changes and potential functions of the seed microbiota during seed development. In this study, we investigated the composition and potential functions of the seed microbiota of rapeseed (Brassica napus). A total of 2496 amplicon sequence variants (ASVs) belonging to 504 genera in 25 phyla were identified, and the seed microbiota of all sampling stages were divided into three groups. The microbiota of flower buds, young pods, and seeds at 20 days after flowering (daf) formed the first group; that of seeds at 30 daf, 40 daf and 50 daf formed the second group; that of mature seeds and parental seeds were clustered into the third group. The functions of seed microbiota were identified by using PICRUSt2, and it was found that the substance metabolism of seed microbiota was correlated with those of the seeds. Finally, sixty-one core ASVs, including several potential human pathogens, were identified, and a member of the seed core microbiota, Sphingomonas endophytica, was isolated from seeds and found to promote seedling growth and enhance resistance against Sclerotinia sclerotiorum, a major pathogen in rapeseed. Our findings provide a novel perspective for understanding the composition and functions of microbiota during seed development and may enhance the efficiency of mining beneficial seed microbes.

Fungal Strains with Identical Genomes Were Found at a Distance of 2000 Kilometers after 40 Years.

Heredity and variation are inherent characteristics of species and are mainly reflected in the stability and variation of the genome; the former is relative, while the latter is continuous. However, whether life has both stable genomes and extremely diverse genomes at the same time is unknown. In this study, we isolated Sclerotinia sclerotiorum strains from sclerotium samples in Quincy, Washington State, USA, and found that four single-sclerotium-isolation strains (PB4, PB273, PB615, and PB623) had almost identical genomes to the reference strain 1980 isolated in the west of Nebraska 40 years ago. The genome of strain PB4 sequenced by the next-generation sequencing (NGS) and Pacific Biosciences (PacBio) sequencing carried only 135 single nucleotide polymorphisms (SNPs) and 18 structural variations (SVs) compared with the genome of strain 1980 and 48 SNPs were distributed on Contig_20. Based on data generated by NGS, three other strains, PB273, PB615, and PB623, had 256, 275, and 262 SNPs, respectively, against strain 1980, which were much less than in strain PB4 (532 SNPs) and none of them occurred on Contig_20, suggesting much closer genomes to strain 1980 than to strain PB4. All other strains from America and China are rich in SNPs with a range of 34,391-77,618 when compared with strain 1980. We also found that there were 39-79 SNPs between strain PB4 and its sexual offspring, 53.1% of which also occurred on Contig_20. Our discoveries show that there are two types of genomes in S. sclerotiorum, one is very stable and the other tends to change constantly. Investigating the mechanism of such genome stability will enhance our understanding of heredity and variation.

Codon Usage Provides Insights into the Adaptive Evolution of Mycoviruses in Their Associated Fungi Host.

Codon usage bias (CUB) could reflect co-evolutionary changes between viruses and hosts in contrast to plant and animal viruses, and the systematic analysis of codon usage among the mycoviruses that infect plant pathogenic fungi is limited. We performed an extensive analysis of codon usage patterns among 98 characterized RNA mycoviruses from eight phytopathogenic fungi. The GC and GC3s contents of mycoviruses have a wide variation from 29.35% to 64.62% and 24.32% to 97.13%, respectively. Mycoviral CUB is weak, and natural selection plays a major role in the formation of mycoviral codon usage pattern. In this study, we demonstrated that the codon usage of mycoviruses is similar to that of some host genes, especially those involved in RNA biosynthetic process and transcription, suggesting that CUB is a potential evolutionary mechanism that mycoviruses adapt to in their hosts.

Codon usage and protein length-dependent feedback from translation elongation regulates translation initiation and elongation speed.

Essential cellular functions require efficient production of many large proteins but synthesis of large proteins encounters many obstacles in cells. Translational control is mostly known to be regulated at the initiation step. Whether translation elongation process can feedback to regulate initiation efficiency is unclear. Codon usage bias, a universal feature of all genomes, plays an important role in determining gene expression levels. Here, we discovered that there is a conserved but codon usage-dependent genome-wide negative correlation between protein abundance and CDS length. The codon usage effects on protein expression and ribosome flux on mRNAs are influenced by CDS length; optimal codon usage preferentially promotes production of large proteins. Translation of mRNAs with long CDS and non-optimal codon usage preferentially induces phosphorylation of initiation factor eIF2α, which inhibits translation initiation efficiency. Deletion of the eIF2α kinase CPC-3 (GCN2 homolog) in Neurospora preferentially up-regulates large proteins encoded by non-optimal codons. Surprisingly, CPC-3 also inhibits translation elongation rate in a codon usage and CDS length-dependent manner, resulting in slow elongation rates for long CDS mRNAs. Together, these results revealed a codon usage and CDS length-dependent feedback mechanism from translation elongation to regulate both translation initiation and elongation kinetics.

Effects of codon usage on gene expression are promoter context dependent.

Codon usage bias is a universal feature of all genomes. Although codon usage has been shown to regulate mRNA and protein levels by influencing mRNA decay and transcription in eukaryotes, little or no genome-wide correlations between codon usage and mRNA levels are detected in mammalian cells, raising doubt on the significance of codon usage effect on gene expression. Here we show that gene-specific regulation reduces the genome-wide codon usage and mRNA correlations: Constitutively expressed genes exhibit much higher genome-wide correlations than differentially expressed genes from fungi to human cells. Using Drosophila S2 cells as a model system, we showed that the effect of codon usage on mRNA expression level is promoter-dependent. Regions downstream of the core promoters of differentially expressed genes can repress the codon usage effects on mRNA expression. An element in the Hsp70 promoter was identified to be necessary and sufficient for this inhibitory effect. The promoter-dependent codon usage effects on mRNA levels are regulated at the transcriptional level through modulation of histone modifications, nucleosome densities and premature termination. Together, our results demonstrate that promoters play a major role in determining whether codon usage influences gene expression and further establish the transcription-dependent codon usage effects on gene expression.

Nonoptimal Codon Usage Is Critical for Protein Structure and Function of the Master General Amino Acid Control Regulator CPC-1.

Under amino acid starvation conditions, eukaryotic organisms activate a general amino acid control response. In Neurospora crassa, Cross Pathway Control Protein 1 (CPC-1), the ortholog of the Saccharomyces cerevisiae bZIP transcription factor GCN4, functions as the master regulator of the general amino acid control response. Codon usage biases are a universal feature of eukaryotic genomes and are critical for regulation of gene expression. Although codon usage has also been implicated in the regulation of protein structure and function, genetic evidence supporting this conclusion is very limited. Here, we show that Neurospora cpc-1 has a nonoptimal NNU-rich codon usage profile that contrasts with the strong NNC codon preference in the genome. Although substitution of the cpc-1 NNU codons with synonymous NNC codons elevated CPC-1 expression in Neurospora, it altered the CPC-1 degradation rate and abolished its amino acid starvation-induced protein stabilization. The codon-manipulated CPC-1 protein also exhibited different sensitivity to limited protease digestion. Furthermore, CPC-1 functions in rescuing the cell growth of the cpc-1 deletion mutant and activation of the expression of its target genes were impaired by the synonymous codon changes. Together, these results reveal the critical role of codon usage in regulation of CPC-1 expression and function and establish a genetic example of the importance of codon usage in protein folding.IMPORTANCE The general amino acid control response is critical for adaptation of organisms to amino acid starvation conditions. The preference to use certain synonymous codons is a universal feature of all genomes. Synonymous codon changes were previously thought to be silent mutations. In this study, we showed that the Neurospora cpc-1 gene has an unusual codon usage profile compared to other genes in the genome. We found that codon optimization of the cpc-1 gene without changing its amino acid sequence resulted in elevated CPC-1 expression, an altered protein degradation rate, and impaired protein functions due to changes in protein structure. Together, these results reveal the critical role of synonymous codon usage in regulation of CPC-1 expression and function and establish a genetic example of the importance of codon usage in protein structure.

Adaptation of codon usage to tRNA I34 modification controls translation kinetics and proteome landscape.

Codon usage bias is a universal feature of all genomes and plays an important role in regulating protein expression levels. Modification of adenosine to inosine at the tRNA anticodon wobble position (I34) by adenosine deaminases (ADATs) is observed in all eukaryotes and has been proposed to explain the correlation between codon usage and tRNA pool. However, how the tRNA pool is affected by I34 modification to influence codon usage-dependent gene expression is unclear. Using Neurospora crassa as a model system, by combining molecular, biochemical and bioinformatics analyses, we show that silencing of adat2 expression severely impaired the I34 modification levels for the ADAT-related tRNAs, resulting in major ADAT-related tRNA profile changes and reprogramming of translation elongation kinetics on ADAT-related codons. adat2 silencing also caused genome-wide codon usage-biased ribosome pausing on mRNAs and proteome landscape changes, leading to selective translational repression or induction of different mRNAs. The induced expression of CPC-1, the Neurospora ortholog of yeast GCN4p, mediates the transcriptional response after adat2 silencing and amino acid starvation. Together, our results demonstrate that the tRNA I34 modification by ADAT plays a major role in driving codon usage-biased translation to shape proteome landscape.

MAPKK Inhibitor U0126 Inhibits Plasmodiophora brassicae Development.

Mitogen-activated protein kinase (MAPK) cascades play a central role in cellular growth, proliferation, and survival. MAPK cascade genes have been extensively investigated in model plants, mammals, yeast, and fungi but are not characterized in Plasmodiophora brassicae, which causes clubroot disease in cruciferous plants. Here, we identified 7 PbMAPK, 3 PbMAPKK, and 9 PbMAPKKK genes in the P. brassicae genome. Transcriptional profiling analysis demonstrated that several MAPK, MAPK kinase (MAPKK), and MAPK kinase kinase (MAPKKK) genes were preferentially expressed in three different zoosporic stages. Based on yeast two-hybrid assays, PbMAKKK7 interacted with PbMAKK3 and PbMAKK3 interacted with PbMAK1/PbMAK3. The PbMAKKK7-PbMAKK3-PbMAK1/PbMAK3 cascade may be present in P. brassicae. U0126, a potent and specific inhibitor of MAPKK, could inhibit the germination of P. brassicae resting spores. U0126 was used to treat the resting spores of P. brassicae and coinoculate rapeseed, and was proven to significantly relieve the severity of clubroot symptoms in the host plant and delay the life cycle of P. brassicae. These results suggest that MAPK signaling pathways may play important roles in P. brassicae growth, development, and pathogenicity.

A Small Secreted Virulence-Related Protein Is Essential for the Necrotrophic Interactions of Sclerotinia sclerotiorum with Its Host Plants.

Small, secreted proteins have been found to play crucial roles in interactions between biotrophic/hemi-biotrophic pathogens and plants. However, little is known about the roles of these proteins produced by broad host-range necrotrophic phytopathogens during infection. Here, we report that a cysteine-rich, small protein SsSSVP1 in the necrotrophic phytopathogen Sclerotinia sclerotiorum was experimentally confirmed to be a secreted protein, and the secretion of SsSSVP1 from hyphae was followed by internalization and cell-to-cell movement independent of a pathogen in host cells. SsSSVP1∆SP could induce significant plant cell death and targeted silencing of SsSSVP1 resulted in a significant reduction in virulence. Through yeast two-hybrid (Y2H), coimmunoprecipitation (co-IP) and bimolecular fluorescence complementation (BiFC) assays, we demonstrated that SsSSVP1∆SP interacted with QCR8, a subunit of the cytochrome b-c1 complex of mitochondrial respiratory chain in plants. Double site-directed mutagenesis of two cysteine residues (C38 and C44) in SsSSVP1∆SP had significant effects on its homo-dimer formation, SsSSVP1∆SP-QCR8 interaction and plant cell death induction, indicating that partial cysteine residues surely play crucial roles in maintaining the structure and function of SsSSVP1. Co-localization and BiFC assays showed that SsSSVP1∆SP might hijack QCR8 to cytoplasm before QCR8 targeting into mitochondria, thereby disturbing its subcellular localization in plant cells. Furthermore, virus induced gene silencing (VIGS) of QCR8 in tobacco caused plant abnormal development and cell death, indicating the cell death induced by SsSSVP1∆SP might be caused by the SsSSVP1∆SP-QCR8 interaction, which had disturbed the QCR8 subcellular localization and hence disabled its biological functions. These results suggest that SsSSVP1 is a potential effector which may manipulate plant energy metabolism to facilitate the infection of S. sclerotiorum. Our findings indicate novel roles of small secreted proteins in the interactions between host-non-specific necrotrophic fungi and plants, and highlight the significance to illuminate the pathogenic mechanisms of this type of interaction.

Comparative genomic and transcriptional analyses of the carbohydrate-active enzymes and secretomes of phytopathogenic fungi reveal their significant roles during infection and development.

Our comparative genomic analysis showed that the numbers of plant cell wall (PCW)- and fungal cell wall (FCW)-degradation-associated carbohydrate-active enzymes (CAZymes) in necrotrophic and hemibiotrophic fungi are significantly larger than that in most biotrophic fungi. However, our transcriptional analyses of CAZyme-encoding genes in Melampsora larici-populina, Puccinia graminis and Sclerotinia sclerotiorum showed that many genes encoding PCW- and FCW-degradation-associated CAZymes were significantly up-regulated during the infection of both necrotrophic fungi and biotrophic fungi, indicating an existence of a universal mechanism underlying PCW degradation and FCW reorganization or modification, which are both intimately involved in necrotrophic and biotrophic fungal infection. Furthermore, our results showed that the FCW reorganization or modification was also related to the fungal development. Additionally, our transcriptional analysis of the secretome of S. sclerotiorum showed that many secreted protein-encoding genes were dramatically induced during infection. Among them, a small, cysteine-rich protein SsCVNH was experimentally confirmed to be essential for the virulence and sclerotial development, indicating that the small secreted proteins might also play crucial roles as potential effectors in host-non-specific necrotrophic fungi.

A "footprint" of plant carbon fixation cycle functions during the development of a heterotrophic fungus.

Carbon fixation pathway of plants (CFPP) in photosynthesis converts solar energy to biomass, bio-products and biofuel. Intriguingly, a large number of heterotrophic fungi also possess enzymes functionally associated with CFPP, raising the questions about their roles in fungal development and in evolution. Here, we report on the presence of 17 CFPP associated enzymes (ten in Calvin-Benson-Basham reductive pentose phosphate pathway and seven in C4-dicarboxylic acid cycle) in the genome of Sclerotinia sclerotiorum, a heterotrophic phytopathogenic fungus, and only two unique enzymes: ribulose-1, 5-bisphosphate carboxylase-oxygenase (Rubisco) and phosphoribulokinase (PRK) were absent. This data suggested an incomplete CFPP-like pathway (CLP) in fungi. Functional profile analysis demonstrated that the activity of the incomplete CLP was dramatically regulated during different developmental stages of S. sclerotiorum. Subsequent experiments confirmed that many of them were essential to the virulence and/or sclerotial formation. Most of the CLP associated genes are conserved in fungi. Phylogenetic analysis showed that many of them have undergone gene duplication, gene acquisition or loss and functional diversification in evolutionary history. These findings showed an evolutionary links in the carbon fixation processes of autotrophs and heterotrophs and implicated the functions of related genes were in course of continuous change in different organisms in evolution.

Two Cdc2 Kinase Genes with Distinct Functions in Vegetative and Infectious Hyphae in Fusarium graminearum.

Eukaryotic cell cycle involves a number of protein kinases important for the onset and progression through mitosis, most of which are well characterized in the budding and fission yeasts and conserved in other fungi. However, unlike the model yeast and filamentous fungi that have a single Cdc2 essential for cell cycle progression, the wheat scab fungus Fusarium graminearum contains two CDC2 orthologs. The cdc2A and cdc2B mutants had no obvious defects in growth rate and conidiation but deletion of both of them is lethal, indicating that these two CDC2 orthologs have redundant functions during vegetative growth and asexual reproduction. However, whereas the cdc2B mutant was normal, the cdc2A mutant was significantly reduced in virulence and rarely produced ascospores. Although deletion of CDC2A had no obvious effect on the formation of penetration branches or hyphopodia, the cdc2A mutant was limited in the differentiation and growth of infectious growth in wheat tissues. Therefore, CDC2A plays stage-specific roles in cell cycle regulation during infectious growth and sexual reproduction. Both CDC2A and CDC2B are constitutively expressed but only CDC2A was up-regulated during plant infection and ascosporogenesis. Localization of Cdc2A- GFP to the nucleus but not Cdc2B-GFP was observed in vegetative hyphae, ascospores, and infectious hyphae. Complementation assays with chimeric fusion constructs showed that both the N- and C-terminal regions of Cdc2A are important for its functions in pathogenesis and ascosporogenesis but only the N-terminal region is important for its subcellular localization. Among the Sordariomycetes, only three Fusarium species closely related to F. graminearum have two CDC2 genes. Furthermore, F. graminearum uniquely has two Aurora kinase genes and one additional putative cyclin gene, and its orthologs of CAK1 and other four essential mitotic kinases in the budding yeast are dispensable for viability. Overall, our data indicate that cell cycle regulation is different between vegetative and infectious hyphae in F. graminearum and Cdc2A, possibly by interacting with a stage-specific cyclin, plays a more important role than Cdc2B during ascosporogenesis and plant infection.

The Microbial Opsin Homolog Sop1 is involved in Sclerotinia sclerotiorum Development and Environmental Stress Response.

Microbial opsins play a crucial role in responses to various environmental signals. Here, we report that the microbial opsin homolog gene sop1 from the necrotrophic phytopathogenic fungus Sclerotinia sclerotiorum was dramatically up-regulated during infection and sclerotial development compared with the vegetative growth stage. Further, study showed that sop1 was essential for growth, sclerotial development and full virulence of S. sclerotiorum. Sop1-silenced transformants were more sensitive to high salt stress, fungicides and high osmotic stress. However, they were more tolerant to oxidative stress compared with the wild-type strain, suggesting that sop1 is involved in different stress responses and fungicide resistance, which plays a role in the environmental adaptability of S. sclerotiorum. Furthermore, a Delta blast search showed that microbial opsins are absent from the genomes of animals and most higher plants, indicating that sop1 is a potential drug target for disease control of S. sclerotiorum.