Molecular characterization of cross-kingdom RNA interference in Botrytis cinerea by tomato small RNAs.

Previous studies have suggested that plants can modulate gene expression in pathogenic fungi by producing small RNAs (sRNAs) that can be translocated into the fungus and mediate gene silencing, which may interfere with the infection mechanism of the intruder. We sequenced sRNAs and mRNAs in early phases of the Solanum lycopersicum (tomato)-Botrytis cinerea interaction and examined the potential of plant sRNAs to silence their predicted mRNA targets in the fungus. Almost a million unique plant sRNAs were identified that could potentially target 97% of all fungal genes. We selected three fungal genes for detailed RT-qPCR analysis of the correlation between the abundance of specific plant sRNAs and their target mRNAs in the fungus. The fungal Bcspl1 gene, which had been reported to be important for the fungal virulence, showed transient down-regulation around 20 hours post inoculation and contained a unique target site for a single plant sRNA that was present at high levels. In order to study the functionality of this plant sRNA in reducing the Bcspl1 transcript level, we generated a fungal mutant that contained a 5-nucleotide substitution that would abolish the interaction between the transcript and the sRNA without changing the encoded protein sequence. The level of the mutant Bcspl1 transcript showed a transient decrease similar to wild type transcript, indicating that the tomato sRNA was not responsible for the downregulation of the Bcspl1 transcript. The virulence of the Bcspl1 target site mutant was identical to the wild type fungus.

Molecular characterization reveals no functional evidence for naturally occurring cross-kingdom RNA interference in the early stages of Botrytis cinerea-tomato interaction.

Plant immune responses are triggered during the interaction with pathogens. The fungus Botrytis cinerea has previously been reported to use small RNAs (sRNAs) as effector molecules capable of interfering with the host immune response. Conversely, a host plant produces sRNAs that may interfere with the infection mechanism of an intruder. We used high-throughput sequencing to identify sRNAs produced by B. cinerea and Solanum lycopersicum (tomato) during early phases of interaction and to examine the expression of their predicted mRNA targets in the other organism. A total of 7042 B. cinerea sRNAs were predicted to target 3185 mRNAs in tomato. Of the predicted tomato target genes, 163 were indeed transcriptionally down-regulated during the early phase of infection. Several experiments were performed to study a causal relation between the production of B. cinerea sRNAs and the down-regulation of predicted target genes in tomato. We generated B. cinerea mutants in which a transposon region was deleted that is the source of c.10% of the fungal sRNAs. Furthermore, mutants were generated in which both Dicer-like genes (Bcdcl1 and Bcdcl2) were deleted and these displayed a >99% reduction of transposon-derived sRNA production. Neither of these mutants was significantly reduced in virulence on any plant species tested. Our results reveal no evidence for any detectable role of B. cinerea sRNAs in the virulence of the fungus.

Comparative analysis of Penicillium genomes reveals the absence of a specific genetic basis for biocontrol in Penicillium rubens strain 212.

Penicillium rubens strain 212 (PO212) is a filamentous fungus belonging to the division Ascomycete. PO212 acts as an effective biocontrol agent against several pathogens in a variety of horticultural crops including Fusarium oxysporum f.sp. lycopersici, causing vascular wilt disease in tomato plants. We assembled draft genomes of two P. rubens strains, the biocontrol agent PO212 and the soil isolate S27, which lacks biocontrol activity. We also performed comparative analyses of the genomic sequence of PO212 with that of the other P. rubens and P. chrysogenum strains. This is the first Penicillium strain with biocontrol activity whose genome has been sequenced and compared. PO212 genome size is 2,982 Mb, which is currently organized into 65 scaffolds and a total of 10,164 predicted Open Reading Frames (ORFs). Sequencing confirmed that PO212 belongs to P. rubens clade. The comparative analysis of the PO212 genome with the genomes of other P. rubens and Penicillium chrysogenum strains available in databases showed strong conservation among genomes, but a correlation was not found between these genomic data and the biocontrol phenotype displayed by PO212. Finally, the comparative analysis between PO212 and S27 genomes showed high sequence conservation and a low number of variations mainly located in ORF regions. These differences found in coding regions between PO212 and S27 genomes can explain neither the biocontrol activity of PO212 nor the absence of such activity in S27, opening a possible avenue toward transcriptomic and epigenetic studies that may shed light on this mechanism for fighting plant diseases caused by fungal pathogens. The genome sequences described in this study provide a useful novel resource for future research into the biology, ecology, and evolution of biological control agents.

The Non-Pathogenic Fusarium oxysporum Fo47 Induces Distinct Responses in Two Closely Related Solanaceae Plants against the Pathogen Verticillium dahliae.

The non-pathogenic Fusarium oxysporum Fo47 is able to protect Capsicum annuum (pepper) but not in Solanum lycopersicum (tomato) against the pathogen Verticillium dahliae. Transcriptomics of the plant during the interaction with Fo47 shows the induction of distinct set of genes in pepper and tomato. The number of differentially expressed (DE) genes in pepper (231 DE genes) is greater than the number of DE genes in tomato (39 DE genes) at 2 days after the treatment with Fo47. Ethylene related genes were present among the DE genes in both plants, and the up-regulation of ethylene biosynthetic genes was observed to be triggered during the interaction of both plants with Fo47. The treatment with MCP (1-Methylcyclopropene, an ethylene-competitive inhibitor) reduced the Fo47 protection in pepper against Verticillium dahliae. Intriguingly, Fo47 was able to protect the ethylene-insensitive tomato mutant Never-ripe (Nr) against Verticillium dahliae, but not the tomato wilt type cv Pearson. Overall, ethylene is shown to be an important player in the response to Fo47, but its role depends on the host species.

Comparative genomics of plant pathogenic Botrytis species with distinct host specificity.

BACKGROUND: Fungi of the genus Botrytis (presently containing ~ 35 species) are able to infect more than 1400 different plant species and cause losses in a wide range of crops of economic importance. The best studied species is B. cinerea, which has a broad host range and is one of the best studied necrotrophic plant pathogenic fungi. Most other Botrytis spp. have a narrow host range and have been studied in less detail. To characterize genomic variation among different representatives of Botrytis spp., we sequenced and annotated the draft genomes of nine Botrytis species: B. calthae, B. convoluta, B. elliptica, B. galanthina, B. hyacinthi, B. narcissicola, B. paeoniae, B. porri and B. tulipae. RESULTS: Bioinformatics and comparative genomics tools were applied to determine a core of 7668 shared protein families in all Botrytis species, which grouped them in two distinct phylogenetic clades. The secretome of all nine Botrytis spp. was similar in number (ranging from 716 to 784 predicted proteins). A detailed analysis of the molecular functions of the secretome revealed that shared activities were highly similar. Orthologs to effectors functionally studied in B. cinerea were also present in the other Botrytis species. A complex pattern of presence/absence of secondary metabolite biosynthetic key enzymes was observed. CONCLUSIONS: Comparative genomics of Botrytis show that overall, species share the main signatures and protein families in the secreted proteins, and of known effectors. Our study provides leads to study host range determinants in the genus Botrytis and provides a stepping stone to elucidate the roles of effector candidates in the infection process of these species.

Vanillyl nonanoate induces systemic resistance and lignification in pepper plants.

The treatment of the cotyledons of pepper plants with vanillyl nonanoate (VNT), a synthetic capsinoid similar to capsiate, protected systemically the plant against a root pathogen (the hemibiotrophic oomycete Phytophthora capsici) and an aerial pathogen (the necrotrophic fungus Botrytis cinerea). VNT treatment reduced both the symptoms and the colonization by these pathogens. VNT induced systemically two PR (Pathogenesis-related) genes and a gene involved in phytoalexin biosynthesis. VNT also induced systemically the reinforcement of cell walls with lignin both in the roots and the leaves. The increase in lignin was correlated with an increase in peroxidase gene expression and activity, pointing to the role of this enzyme in lignin polymerization. The results suggest that VNT induces systemic resistance at least in part by means of lignification.

Many Shades of Grey in Botrytis-Host Plant Interactions.

The grey mould Botrytis cinerea causes disease in more than 1000 plant species, including important crops. The interaction between Botrytis and its (potential) hosts is determined by quantitative susceptibility and virulence traits in both interacting partners, resulting in a greyscale of disease outcomes. Fungal infection was long thought to rely mainly on its capacity to kill the host plant and degrade plant tissue. Recent research has revealed that Botrytis exploits two crucial biological processes in host plants for its own success. We highlight recent findings that illustrate that the interactions between Botrytis and its host plants are subtle and we discuss the molecular and cellular mechanisms controlling the many shades of grey during these interactions.

Functional Analysis of Mating Type Genes and Transcriptome Analysis during Fruiting Body Development of Botrytis cinerea.

Botrytis cinerea is a plant-pathogenic fungus producing apothecia as sexual fruiting bodies. To study the function of mating type (MAT) genes, single-gene deletion mutants were generated in both genes of the MAT1-1 locus and both genes of the MAT1-2 locus. Deletion mutants in two MAT genes were entirely sterile, while mutants in the other two MAT genes were able to develop stipes but never formed an apothecial disk. Little was known about the reprogramming of gene expression during apothecium development. We analyzed transcriptomes of sclerotia, three stages of apothecium development (primordia, stipes, and apothecial disks), and ascospores by RNA sequencing. Ten secondary metabolite gene clusters were upregulated at the onset of sexual development and downregulated in ascospores released from apothecia. Notably, more than 3,900 genes were differentially expressed in ascospores compared to mature apothecial disks. Among the genes that were upregulated in ascospores were numerous genes encoding virulence factors, which reveals that ascospores are transcriptionally primed for infection prior to their arrival on a host plant. Strikingly, the massive transcriptional changes at the initiation and completion of the sexual cycle often affected clusters of genes, rather than randomly dispersed genes. Thirty-five clusters of genes were jointly upregulated during the onset of sexual reproduction, while 99 clusters of genes (comprising >900 genes) were jointly downregulated in ascospores. These transcriptional changes coincided with changes in expression of genes encoding enzymes participating in chromatin organization, hinting at the occurrence of massive epigenetic regulation of gene expression during sexual reproduction.IMPORTANCE Fungal fruiting bodies are formed by sexual reproduction. We studied the development of fruiting bodies ("apothecia") of the ubiquitous plant-pathogenic ascomycete Botrytis cinerea The role of mating type genes in apothecium development was investigated by targeted mutation. Two genes are essential for the initiation of sexual development; mutants in these genes are sterile. Two other genes were not essential for development of stipes; however, they were essential for stipes to develop a disk and produce sexual ascospores. We examined gene expression profiles during apothecium development, as well as in ascospores sampled from apothecia. We provide the first study ever of the transcriptome of pure ascospores in a filamentous fungus. The expression of numerous genes involved in plant infection was induced in the ascospores, implying that ascospores are developmentally primed for infection before their release from apothecia.

Wounding induces local resistance but systemic susceptibility to Botrytis cinerea in pepper plants.

Cotyledon wounding in pepper caused the early generation of hydrogen peroxide both locally (cotyledons) and systemically (upper true leaves). However, 72 h later there is a different wound response between local and systemic organs, as shown by resistance to the pathogenic fungus Botrytis cinerea, that increased locally and decreased systemically. Signaling by ethylene and jasmonic acid was assessed by using two inhibitors: 1-methylcyclopropene (MCP, inhibitor of ethylene receptors) and ibuprofen (inhibitor of jasmonate biosynthesis). MCP did not affect the modulation of resistance levels to Botrytis by wounding, ruling out the involvement of ethylene signaling. Ibuprofen did not inhibit wound-induced resistance at the local level, but inhibited wound-induced systemic susceptibility. Moreover, changes of biochemical and structural defenses in response to wounding were studied. Peroxidase activity and the expression of a peroxidase gene (CAPO1) increased locally as a response to wounding, but no changes were observed systemically. Lignin deposition was induced in wounded cotyledons, but was repressed in systemic leaves of wounded plants, whereas soluble phenolics did not change locally and decreased systemically. The expression of two other genes involved in plant defense (CABPR1 and CASC1) was also differentially regulated locally and systemically, pointing to a generalized increase in plant defenses at the local level and a systemic decrease as a response to wounding. Wound-induced defenses at the local level coincided with resistance to the necrotroph fungus B. cinerea, whereas depleted defenses in systemic leaves of wounded plants correlated to induced susceptibility against this pathogen. It may be that the local response acts as a sink of energy resources to mount a defense against pathogens, whereas in systemic organs the resources for defense are lower.