UvHOS3-mediated histone deacetylation is essential for virulence and negatively regulates ustilaginoidin biosynthesis in Ustilaginoidea virens.

Ustilaginoidea virens is the causal agent of rice false smut, which has recently become one of the most important rice diseases worldwide. Ustilaginoidins, a major type of mycotoxins produced in false smut balls, greatly deteriorates grain quality. Histone acetylation and deacetylation are involved in regulating secondary metabolism in fungi. However, little is yet known on the functions of histone deacetylases (HDACs) in virulence and mycotoxin biosynthesis in U. virens. Here, we characterized the functions of the HDAC UvHOS3 in U. virens. The ΔUvhos3 deletion mutant exhibited the phenotypes of retarded growth, increased mycelial branches and reduced conidiation and virulence. The ΔUvhos3 mutants were more sensitive to sorbitol, sodium dodecyl sulphate and oxidative stress/H2 O2 . ΔUvhos3 generated significantly more ustilaginoidins. RNA-Seq and metabolomics analyses also revealed that UvHOS3 is a key negative player in regulating secondary metabolism, especially mycotoxin biosynthesis. Notably, UvHOS3 mediates deacetylation of H3 and H4 at H3K9, H3K18, H3K27 and H4K8 residues. Chromatin immunoprecipitation assays indicated that UvHOS3 regulates mycotoxin biosynthesis, particularly for ustilaginoidin and sorbicillinoid production, by modulating the acetylation level of H3K18. Collectively, this study deepens the understanding of molecular mechanisms of the HDAC UvHOS3 in regulating virulence and mycotoxin biosynthesis in phytopathogenic fungi.

Histone Deacetylase UvHST2 Is a Global Regulator of Secondary Metabolism in Ustilaginoidea virens.

Ustilaginoidea virens, the causal agent of rice false smut, produces a large amount of mycotoxins, including ustilaginoidins and sorbicillinoids. However, little is known about the regulatory mechanism of mycotoxin biosynthesis inU. virens. Here, we demonstrate that the NAD+-dependent histone deacetylase UvHST2 negatively regulates ustilaginoidin biosynthesis. UvHst2 knockout caused retarded hypha growth and reduced conidiation and pathogenicity inU. virens. Transcriptome analysis revealed that the transcription factor genes, transporter genes, and other tailoring genes in eight biosynthetic gene clusters (BGCs) including ustilaginoidin and sorbicillinoid BGCs were upregulated in ΔUvhst2. Interestingly, the UvHst2 deletion affects alternative splicing. Metabolomics revealed that UvHST2 negatively regulates the biosynthesis of various mycotoxins including ustilaginoidins, sorbicillin, ochratoxin B, zearalenone, and O-M-sterigmatocystin. Combined transcriptome and metabolome analyses uncover that UvHST2 positively regulates pathogenicity but negatively modulates the expression of BGCs involved in secondary metabolism. Collectively, UvHST2 functions as a global regulator of secondary metabolism inU. virens.

An Ustilaginoidea virens glycoside hydrolase 42 protein is an essential virulence factor and elicits plant immunity as a PAMP.

Rice false smut, caused by the ascomycete fungus Ustilaginoidea virens, which infects rice florets before heading, severely threatens rice grain yield and quality worldwide. The U. virens genome encodes a number of glycoside hydrolase (GH) proteins. So far, the functions of these GHs in U. virens are largely unknown. In this study, we identified a GH42 protein secreted by U. virens, named UvGHF1, that exhibits ß-galactosidase activity. UvGHF1 not only functions as an essential virulence factor during U. virens infection, but also serves as a pathogen-associated molecular pattern (PAMP) in Nicotiana benthamiana and rice. The PAMP activity of UvGHF1 is independent of its ß-galactosidase activity. Moreover, UvGHF1 triggers cell death in N. benthamiana in a BAK1-dependent manner. Ectopic expression of UvGHF1 in rice induces pattern-triggered immunity and enhances rice resistance to fungal and bacterial diseases. RNA-seq analysis revealed that UvGHF1 expression in rice not only activates expression of many defence-related genes encoding leucine-rich repeat receptor-like kinases and WRKY and ERF transcription factors, but also induces diterpenoid biosynthesis and phenylpropanoid biosynthesis pathways. Therefore, UvGHF1 contributes to U. virens virulence, but is also recognized by the rice surveillance system to trigger plant immunity.

Genome Assembly and Comparative Analysis of the Egg Parasitoid Wasp Trichogramma dendrolimi Shed Light on the Composition and Evolution of Olfactory Receptors and Venoms.

Trichogramma dendrolimi is one of the most successfully industrialized Trichogramma species used to control agricultural and forestry pests in China. However, the molecular mechanisms underlying its host recognition and parasitism remain largely unknown, partially due to the limited genome information of this parasitoid wasp. Here, we present a high-quality de novo assembly of T. dendrolimi through a combination of Illumina and PacBio sequencing technologies. The final assembly had a length of 215.2 Mb and contains 316 scaffolds with a scaffold N50 size of 1.41 Mb. Repetitive sequences with a length of 63.4 Mb and 12,785 protein-coding genes were identified. Significantly expanded gene families were identified to be involved in the development and regulatory processes, while remarkably contracted gene families were involved in the transport processes in T. dendrolimi. The olfactory and venom-associated genes were identified in T. dendrolimi and 24 other hymenopteran species, using uniform methods combining BLAST and HMM profiling. The identified venom genes of T. dendrolimi were enriched in antioxidant activity, tricarboxylic acid cycle, response to oxidative stress and cell redox homeostasis. Our study provides an important resource for comparative genomics and functional studies to interpret the molecular mechanisms underlying host recognition and parasitism of Trichogramma species.

Correction: Histone H3 gene is not a suitable marker to distinguish Alternaria tenuissima from A. alternata affecting potato.

[This corrects the article DOI: 10.1371/journal.pone.0231961.].

Large-Scale Genome Scanning within Exonic Regions Revealed the Contributions of Selective Sweep Prone Genes to Host Divergence and Adaptation in Magnaporthe oryzae Species Complex.

Magnaporthe oryzae, one of the most notorious plant pathogens in the agronomic ecosystem, causes a destructive rice blast disease around the world. The blast fungus infects wide arrays of cultivated and non-cultivated plants within the Poaceae. Studies have shown that host speciation exerts selection pressure that drives the evolution and divergence of the M. oryzae population. Population genetic relationship deducted by genome-wide single nucleotide polymorphisms showed that M. oryzae differentiation is highly consistent with the host speciation process. In particular, the rice-infecting population of M. oryzae is distinct from populations from other hosts. However, how genome regions prone to host-mediated selection pressures associated with speciation in M. oryzae, especially at a large-scale population level, has not been extensively characterized. Here, we detected strong evidence of sweep selection throughout the genomes of rice and non-rice pathotypes of M. oryzae population using integrated haplotype score (iHS), cross population extended haplotype homozygosity (XPEHH), and cross population composite likelihood ratio (XPCLR) tests. Functional annotation analyses of the genes associated with host-mediated selection pressure showed that 14 pathogenicity-related genes are under positive selection pressure. Additionally, we showed that 17 candidate effector proteins are under positive and divergent selection among the blast fungus population through sweep selection analysis. Specifically, we find that a divergent selective gene, MGG_13871, is experiencing host-directed mutation in two amino acid residues in rice and non-rice infecting populations. These results provide a crucial insight into the impact of selective sweeping on the differentiation of M. oryzae populations and the dynamic influences of genomic regions in promoting host adaptation and speciation among M. oryzae species.

Histone H3 gene is not a suitable marker to distinguish Alternaria tenuissima from A. alternata affecting potato.

Potato Alternaria leaf blight is one of the economically most important disease in potato production worldwide. A recent study reported a quick method to distinguish main Alternaria pathogens A. tenuissima, A. alternata, and A. solani using partial histone H3 gene sequences. Using this method, our collection of 79 isolates from 8 provinces in China were presumably separated into A. tenussima and A. alternata. But in depth morphological and genetic analysis casted doubt on this identification. Culture morphologies of six presumed A. alternata isolates (PresA_alt) and six presumed A. tenuissima isolates (PresA_ten) were not significantly different. PresA_ten isolates also produced conidia in branched chains which supposed to be A. aternata. Phylogenetic analyses were conducted using internal transcribed spacer region (ITS) and five genes commonly used for species identification including glyceraldehyde-3-phosphate dehydrogenase (GPDH), translation elongation factor 1-alpha (TEF1), ß-tubulin, plasma membrane ATPase (ATPase), and calmodulin genes. The results showed that GPDH and TEF1 sequences of PresA_alt and PresA_ten isolates were identical. The 12 isolates did not cluster by presumed species neither by individual or concatenated sequence comparisons. The phylogeny-trait association analysis confirmed that the two group isolates were undistinguishable by those molecular markers. Analysis of histone H3 gene sequences revealed variable intron sequences between PresA_ten and PresA_alt isolates, but the amino acid sequences were identical. Our results indicate that the previously published method to distinguish Alternaria species based on histone H3 gene sequence variation is inaccurate and that the prevalence of A. tenuissima isolates in China was likely overestimated.

The Phytophthora infestans AVR2 Effector Escapes R2 Recognition Through Effector Disordering.

Intrinsic disorder is a common structural characteristic of proteins and a central player in the biochemical processes of species. However, the role of intrinsic disorder in the evolution of plant-pathogen interactions is rarely investigated. Here, we explored the role of intrinsic disorder in the development of the pathogenicity in the RXLR AVR2 effector of Phytophthora infestans. We found AVR2 exhibited high nucleotide diversity generated by point mutation, early-termination, altered start codon, deletion/insertion, and intragenic recombination and is predicted to be an intrinsically disordered protein. AVR2 amino acid sequences conferring a virulent phenotype had a higher disorder tendency in both the N- and C-terminal regions compared with sequences conferring an avirulent phenotype. In addition, we also found virulent AVR2 mutants gained one or two short linear interaction motifs, the critical components of disordered proteins required for protein-protein interactions. Furthermore, virulent AVR2 mutants were predicted to be unstable and have a short protein half-life. Taken together, these results support the notion that intrinsic disorder is important for the effector function of pathogens and demonstrate that SLiM-mediated protein-protein interaction in the C-terminal effector domain might contribute greatly to the evasion of resistance-protein detection in P. infestans.

Lack of gene flow between Phytophthora infestans populations of two neighboring countries with the largest potato production.

Gene flow is an important evolutionary force that enables adaptive responses of plant pathogens in response to changes in the environment and plant disease management strategies. In this study, we made a direct inference concerning gene flow in the Irish famine pathogen Phytophthora infestans between two of its hosts (potato and tomato) as well as between China and India. This was done by comparing sequence characteristics of the eukaryotic translation elongation factor 1 alpha (eEF-1α) gene, generated from 245 P. infestans isolates sampled from two countries and hosts. Consistent with previous results, we found that eEF-1α gene was highly conserved and point mutation was the only mechanism generating any sequence variation. Higher genetic variation was found in the eEF-1α sequences in the P. infestans populations sampled from tomato compared to those sampled from potato. We also found the P. infestans population from India displayed a higher genetic variation in the eEF-1α sequences compared to China. No gene flow was detected between the pathogen populations from the two countries, which is possibly attributed to the geographic barrier caused by Himalaya Plateau and the minimum cross-border trade of potato and tomato products. The implications of these results for a sustainable management of late blight diseases are discussed.

Induced expression of CYP51 associated with difenoconazole resistance in the pathogenic Alternaria sect. on potato in China.

BACKGROUND: Early blight caused by Alternaria spp. is amongst the most important diseases in potato. Demethylation inhibitor (DMI) fungicides are widely used to control the disease but long-term use may decrease its control efficacy due to fungicide resistance. This study investigated the occurrence of difenoconazole resistance in Alternaria spp. and molecular resistant mechanisms. RESULTS: EC50 values of 160 isolates to difenoconazole ranged from 0.026 µg mL-1 to 15.506 µg mL-1 and the frequency of difenoconazole sensitivity formed a non-normal distribution curve with a major and a minor peak. Isolates with EC50 values of 4.121 and 5.461 µg mL-1 were not controlled effectively at fungicide doses of 50 and 100 µg mL-1 . Cross-resistance was observed between DMI fungicides difenoconazole and propiconazole, but not between difenoconazole and other fungicide groups, including boscalid, iprodione, or carbendazim. The CYP51gene was 1673 bp encoding 525 amino acids in length and contained two introns. All sensitive and resistant isolates had the identical amino acid sequence of CYP51, with the exception of one resistant isolate carrying a mutation of R511W. A 6 bp insertion in the upstream region was observed in half of the resistant isolates. In the absence of propiconazole, the relative expression of CYP51 was not significantly different in sensitive and resistant isolates. In the presence of difenoconazole, expression of CYP51 gene was induced significantly in the DMI-resistant isolates but not in the sensitive ones. CONCLUSION: Induced expression of CYP51 in resistant isolates exposed to difenoconazole is an important determinant for DMI resistance in potato pathogens Alternaria sect. © 2019 Society of Chemical Industry.

Europe was a hub for the global spread of potato virus S in the 19th century.

Potato virus S (PVS) is a major plant pathogen that causes considerable losses in global potato production. Knowledge of the evolutionary history and spatio-temporal dynamics of PVS is vital for developing sustainable management schemes. In this study, we investigated the phylodynamics of the virus by analysing 103 nucleotide sequences of the coat protein gene, sampled between 1985 and 2014. Our Bayesian phylogenetic analyses showed that PVS has been evolving at a rate of 3.32 × 10-4 substitutions/site/year (95% credibility interval 1.33 × 10-4-5.58 × 10-4). We dated the crown group to the year 1325 CE (95% credibility interval 762-1743 CE). Our phylogeographic analyses pointed to viral origins in South America and identified multiple migration pathways between Europe and other regions, suggesting that Europe has been a major hub for PVS transmission. The results of our study have potential implications for developing effective strategies for the control of this pathogen.

Host Resistance and Temperature-Dependent Evolution of Aggressiveness in the Plant Pathogen Zymoseptoria tritici.

Understanding how habitat heterogeneity may affect the evolution of plant pathogens is essential to effectively predict new epidemiological landscapes and manage genetic diversity under changing global climatic conditions. In this study, we explore the effects of habitat heterogeneity, as determined by variation in host resistance and local temperature, on the evolution of Zymoseptoria tritici by comparing the aggressiveness development of five Z. tritici populations originated from different parts of the world on two wheat cultivars varying in resistance to the pathogen. Our results show that host resistance plays an important role in the evolution of Z. tritici. The pathogen was under weak, constraining selection on a host with quantitative resistance but under a stronger, directional selection on a susceptible host. This difference is consistent with theoretical expectations that suggest that quantitative resistance may slow down the evolution of pathogens and therefore be more durable. Our results also show that local temperature interacts with host resistance in influencing the evolution of the pathogen. When infecting a susceptible host, aggressiveness development of Z. tritici was negatively correlated to temperatures of the original collection sites, suggesting a trade-off between the pathogen's abilities of adapting to higher temperature and causing disease and global warming may have a negative effect on the evolution of pathogens. The finding that no such relationship was detected when the pathogen infected the partially resistant cultivars indicates the evolution of pathogens in quantitatively resistant hosts is less influenced by environments than in susceptible hosts.

Population genetic analysis reveals cryptic sex in the phytopathogenic fungus Alternaria alternata.

Reproductive mode can impact population genetic dynamics and evolutionary landscape of plant pathogens as well as on disease epidemiology and management. In this study, we monitored the spatial dynamics and mating type idiomorphs in ~700 Alternaria alternata isolates sampled from the main potato production areas in China to infer the mating system of potato early blight. Consistent with the expectation of asexual species, identical genotypes were recovered from different locations separated by hundreds of kilometers of geographic distance and spanned across many years. However, high genotype diversity, equal MAT1-1 and MAT1-2 frequencies within and among populations, no genetic differentiation and phylogenetic association between two mating types, combined with random association amongst neutral markers in some field populations, suggested that sexual reproduction may also play an important role in the epidemics and evolution of the pathogen in at least half of the populations assayed despite the fact that no teleomorphs have been observed yet naturally or artificially. Our results indicated that A. alternata may adopt an epidemic mode of reproduction by combining many cycles of asexual propagation with fewer cycles of sexual reproduction, facilitating its adaptation to changing environments and making the disease management on potato fields even more difficult.