Project description:Powdery mildew diseases are a major phytosanitary issue causing important yield and economic losses in agronomic, horticultural and ornamental crops. Powdery mildew fungi are obligate biotrophic parasites unable to grow on culture media, a fact that has significantly limited their genetic manipulation. In this work, we report a protocol based on the electroporation of fungal conidia, for the transient transformation of Podosphaera fusca (synonym Podosphaera xanthii), the main causal agent of cucurbit powdery mildew.To introduce DNA into P. xanthii conidia, we applied two square-wave pulses of 1.7 kV for 1 ms with an interval of 5 s. We tested these conditions with several plasmids bearing as selective markers hygromycin B resistance (hph), carbendazim resistance (TUB2) or GFP (gfp) under control of endogenous regulatory elements from Aspergillus nidulans, Neurospora crassa or P. xanthii to drive their expression. An in planta selection procedure using the MBC fungicide carbendazim permitted the propagation of transformants onto zucchini cotyledons and avoided the phytotoxicity associated with hygromycin B.This is the first report on the transformation of P. xanthii and the transformation of powdery mildew fungi using electroporation. Although the transformants are transient, this represents a feasible method for the genetic manipulation of this important group of plant pathogens.
Project description:Podosphaera xanthii is the main causal agent of cucurbit powdery mildew in Southern Italy. Illumina sequencing of mRNA from two P. xanthii isolates of opposite mating types (MAT1-1 and MAT1-2) and their sexual cross was used to obtain a detailed de novo Trinity-based assembly of the transcriptome of the fungus. Over 60 million of high-quality paired-end reads were obtained and assembled into 71,095 contigs corresponding to putative transcripts that were functionally annotated. More than 55% of the assembled transcripts (40,221 contigs) had a significant hit in BLASTx search and included sequences related to sexual compatibility and reproduction, as well as several classes of transposable elements and putative mycoviruses. The availability of these new transcriptomic data and investigations on potential source of genetic variation in P. xanthii will promote new insights on the pathogen and its interactions with host plants and associated microbiome.
Project description:Powdery mildew (PM) disease, caused by the obligate biotrophic fungal pathogen Podosphaera xanthii, is the most reported and destructive disease on cultivated Cucurbita species all over the world. Recently, the appearance of highly aggressive P. xanthii isolates has led to PM outbreaks even in resistant crops, making disease management a very difficult task. To challenge this, breeders rely on genetic characteristics for PM control. Analysis of commercially available intermediate resistance courgette (Cucurbita pepo L. var. cylindrica) varieties using cytological, molecular, and biochemical approaches showed that the plants were under a primed state and induced systemic acquired resistance (SAR) responses, exhibiting enhanced callose production, upregulation of salicylic acid (SA) defense signaling pathway genes, and accumulation of SA and defense metabolites. Additionally, the intermediate resistant varieties showed an altered epigenetic landscape in histone marks that affect transcriptional activation. We demonstrated that courgette plants had enriched H3K4me3 marks on SA-BINDING PROTEIN 2 and YODA (YDA) genes of the Pm-0 interval introgression, a genomic region that confers resistant to Cucurbits against P. xanthii. The open chromatin of SA-BINDING PROTEIN 2 and YDA genes was consistent with genes' differential expression, induced SA pathway, altered stomata characteristics, and activated SAR responses. These findings demonstrate that the altered epigenetic landscape of the intermediate resistant varieties modulates the activation of SA-BINDING PROTEIN 2 and YDA genes leading to induced gene transcription that primes courgette plants.