The necrotrophic fungal plant pathogen Sclerotinia sclerotiorum is responsible for substantial global crop losses annually resulting in localized food insecurity and loss of livelihood. Understanding the basis of this broad-host-range and aggressive pathogenicity is hampered by the quantitative nature of both host resistance and pathogen virulence. To improve this understanding, methods for efficient functional gene characterization that build upon the existing complete S. sclerotiorum genome sequence are needed. Here, we report on the development of a clustered regularly interspaced short palindromic repeat (CRISPR)–CRISPR-associated protein 9 (CRISPR-Cas9)-mediated strategy for creating gene disruption mutants and the application of this technique for exploring roles of known and hypothesized virulence factors. A key finding of this research is that transformation with a circular plasmid encoding Cas9, target single guide RNA (sgRNA), and a selectable marker resulted in a high frequency of targeted, insertional gene mutation. We observed that 100% of the mutants integrated large rearranged segments of the transforming plasmid at the target site facilitated by the nonhomologous end joining (NHEJ) repair pathway. This result was confirmed in multiple target sites within the same gene in three independent wild-type isolates of S. sclerotiorum and in a second independent gene. Targeting the previously characterized Ssoah1 gene allowed us to confirm the loss-of-function nature of the CRISPR-Cas9-mediated mutants and explore new aspects of the mutant phenotype. Applying this technology to create mutations in a second previously uncharacterized gene allowed us to determine the requirement for melanin accumulation in infection structure development and function. IMPORTANCE Fungi that cause plant diseases by rotting or blighting host tissue with limited specificity remain among the most difficult to control. This is largely due to the quantitative nature of host resistance and a limited understanding of fungal pathogenicity. A mechanistic understanding of pathogenicity requires the ability to manipulate candidate virulence genes to test hypotheses regarding their roles in disease development. Sclerotinia sclerotiorum is among the most notorious of these so-called broad-host-range necrotrophic plant pathogens. The work described here provides a new method for rapidly constructing gene disruption vectors to create gene mutations with high efficiency compared with existing methods. Applying this method to characterize gene functions in S. sclerotiorum , we confirm the requirement for oxalic acid production as a virulence factor in multiple isolates of the fungus and demonstrate that melanin accumulation is not required for infection. Using this approach, the pace of functional gene characterization and the understanding of pathogenicity and related disease resistance will increase.

Summary: The sclerotium, a multicellular structure composed of the compact aggregation of vegetative hyphae, is critical for the long‐term survival and sexual reproduction of the plant‐pathogenic fungus Sclerotinia sclerotiorum . The development and carpogenic germination of sclerotia are regulated by integrating signals from both environmental and endogenous processes. Here, we report the regulatory functions of the S. sclerotiorum GATA‐type IVb zinc‐finger transcription factor SsNsd1 in these processes. SsNsd1 is orthologous to the Aspergillus nidulans NsdD (never in sexual development) and the Neurospora crassa SUB‐1 (submerged protoperithecia‐1) proteins. Ssnsd1 gene transcript accumulation remains relatively low, but variable, during vegetative mycelial growth and multicellular development. Ssnsd1 deletion mutants (Δ nsd1 ‐KOs) produce phialides and phialospores (spermatia) excessively in vegetative hyphae and promiscuously within the interior medulla of sclerotia. In contrast, phialospore development occurs only on the sclerotium surface in the wild‐type. Loss of SsNsd1 function affects sclerotium structural integrity and disrupts ascogonia formation during conditioning for carpogenic germination. As a consequence, apothecium development is abolished. The Ssnsd1 deletion mutants are also defective in the transition from hyphae to compound appressorium formation, resulting in a loss of pathogenicity on unwounded hosts. In sum, our results demonstrate that SsNsd1 functions in a regulatory role similar to its ascomycete orthologues in regulating sexual and asexual development. Further, SsNsd1 appears to have evolved as a regulator of pre‐penetration infectious development required for the successful infection of its many hosts.

The ZmCBF3 gene is a member of AP2/ERF transcription factor family, which is a large family of plant-specific transcription factors that share a well-conserved DNA-binding domain. To understand the regulatory mechanism of ZmCBF3 gene expression, we isolated and characterized the ZmCBF3 promoter (PZmCBF3). Three deletion fragments of PZmCBF3 were generated, C1–C3, from the translation start codon at position −1079, −638, and −234, and fused to the GUS reporter gene. Each deletion construct was analyzed by Agrobacterium-mediated stable transformation and expression in Arabidopsis thaliana. GUS expression assays indicated that the PZmCBF3 exhibited root-specific expression activity. A 234-bp fragment upstream of the ZmCBF3 gene conferred a high level of GUS activity in Arabidopsis. Some cis-acting elements involved in the down-regulation of gene expression were detected in the promoter, encompassing positions −1079 to −234. PZmCBF3 was activated by cold stress. The MYCCONSENSUSAT elements (CANNTG) were responsible for the ability of PZmCBF3 to respond to cold stress. The results of the present study suggest that PZmCBF3 might play a role in cold tolerance in maize.

Magnaporthe oryzae is a fungal pathogen that causes rice blast, a highly destructive disease. In the present study, the bacteria strain GS-1 was isolated from the rhizosphere soil of ginseng and identified as Bacillus velezensis through 16S rRNA gene sequencing, whole genome assembly, and average nucleotide identity analysis. B. velezensis strain GS-1 exhibited significant antagonistic activity to several plant fungal pathogens. Through whole genome sequencing, 92 Carbohydrate-Active Enzymes and 13 gene clusters that encoded for secondary metabolites were identified. In addition, strain GS-1 was able to produce the lipopeptide compounds, surfactin, fengycin, and plantazolicin. The inhibitory effects of lipopeptide compounds on M. oryzae were confirmed, and the antagonistic mechanism was explored using transcriptomics and metabolomics analysis. Differential expressed genes (DEGs) and differential accumulated metabolites (DAMs) revealed that the inhibition of M. oryzae by lipopeptide produced by GS-1 downregulated the expression of genes involved in amino acid metabolism, sugar metabolism, oxidative phosphorylation, and autophagy. These results may explain why GS-1 has antagonistic activity to fungal pathogens and revealed the mechanisms underlying the inhibitory effects of lipopeptides produced by GS-1 on fungal growth, which may provide a theoretical basis for the potential application of B. velezensis GS-1 in future plant protection.