Genetic alteration of UDP‐rhamnose metabolism in Botrytis cinerea leads to the accumulation of UDP‐KDG that adversely affects development and pathogenicity

Abstract

Botrytis cinerea is a model plant-pathogenic fungus that causes grey mould and rot diseases in a wide range of agriculturally important crops. A previous study has identified two enzymes and corresponding genes (bcdh, bcer) that are involved in the biochemical transformation of uridine diphosphate (UDP)-glucose, the major fungal wall nucleotide sugar precursor, to UDP-rhamnose. We report here that deletion of bcdh, the first biosynthetic gene in the metabolic pathway, or of bcer, the second gene in the pathway, abolishes the production of rhamnose-containing glycans in these mutant strains. Deletion of bcdh or double deletion of both bcdh and bcer has no apparent effect on fungal development or pathogenicity. Interestingly, deletion of the bcer gene alone adversely affects fungal development, giving rise to altered hyphal growth and morphology, as well as reduced sporulation, sclerotia production and virulence. Treatments with wall stressors suggest the alteration of cell wall integrity. Analysis of nucleotide sugars reveals the accumulation of the UDP-rhamnose pathway intermediate UDP-4-keto-6-deoxy-glucose (UDP-KDG) in hyphae of the Δbcer strain. UDP-KDG could not be detected in hyphae of the wild-type strain, indicating fast conversion to UDP-rhamnose by the BcEr enzyme. The correlation between high UDP-KDG and modified cell wall and developmental defects raises the possibility that high levels of UDP-KDG result in deleterious effects on cell wall composition, and hence on virulence. This is the first report demonstrating that the accumulation of a minor nucleotide sugar intermediate has such a profound and adverse effect on a fungus. The ability to identify molecules that inhibit Er (also known as NRS/ER) enzymes or mimic UDP-KDG may lead to the development of new antifungal drugs.

BcCFEM1, a CFEM Domain-Containing Protein with Putative GPI-Anchored Site, Is Involved in Pathogenicity, Conidial Production, and Stress Tolerance in Botrytis cinerea

“…Primers were designed across or flanking an intron (Supplementary Table S1 ). The relative expression of B. cinerea Bcgpdh ( BC1G_05277 ) gene ( Ma et al, 2017 ) and P. vulgaris Actin-11 (GenBank: EH040443.1) gene ( Oliveira et al, 2015 ) were respectively used as reference genes for normalizing the RNA sample. For each gene, qRT-PCR assays were respectively repeated at least twice, with each repetition having three independent replicates.…”

BcXYG1, a Secreted Xyloglucanase from Botrytis cinerea, Triggers Both Cell Death and Plant Immune Responses

“…Oligonucleotides used for plasmid construction are described in Supplemental Table S6. The BcXYG1 replacement construct was generated as described (Ma et al, 2017a). The 59 (538 bp) and 39 (534 bp) flanks of the BcXYG1 open reading frame were amplified by PCR from genomic DNA of the wild-type strain B05.10, and the fragments were cloned into the upstream and downstream regions, respectively, of the hph cassette using the Gibson Assembly Master Mix kit (New England Biolabs).…”

“…Transformation of B. cinerea was performed as described previously (Ma et al, 2017a). The following transgenic strains were produced: Dxyg1 (deletion of BcXYG1), OEXYG1 (overexpression of an HA-tagged native form of BcXYG1), and OEMXYG1 (overexpression of an HA-tagged mutated form of BcXYG1 that shows no enzymatic activity).…”

“…Colony morphology and sclerotia formation were monitored after 7 and 15 d in light-and dark-incubated cultures, respectively. For the stress tolerance assay, examined strains were inoculated onto PDA plates containing 1 M NaCl, 1 M sorbitol, 0.3 mg mL 21 Calcofluor White, 0.5 mg mL 21 Congo Red, and 0.02% SDS, as described previously (Ma et al, 2017a).…”

The Subtilisin-Like Protease Bcser2 Affects the Sclerotial Formation, Conidiation and Virulence of Botrytis cinerea

“…Homokaryotic strains of transformants were obtained by single spore isolation which was performed following a previously described protocol with some modifications [58,59]. Conidia of heterokaryotic transformants developed on PDA plates containing hygromycin B or G418 (75 µg/mL) were harvested and then diluted with sterile water, and 20 to 50 spores were spread on selection medium supplemented with hygromycin B or G418 (100 µg/mL).…”