The haustorial transcriptome of the cucurbit pathogen Podosphaera xanthii reveals new insights into the biotrophy and pathogenesis of powdery mildew fungi

Abstract

Background Podosphaera xanthii is the main causal agent of powdery mildew disease in cucurbits and is responsible for important yield losses in these crops worldwide. Powdery mildew fungi are obligate biotrophs. In these parasites, biotrophy is determined by the presence of haustoria, which are specialized structures of parasitism developed by these fungi for the acquisition of nutrients and the delivery of effectors. Detailed molecular studies of powdery mildew haustoria are scarce due mainly to difficulties in their isolation. Therefore, their analysis is considered an important challenge for powdery mildew research. The aim of this work was to gain insights into powdery mildew biology by analysing the haustorial transcriptome of P. xanthii . Results Prior to RNA isolation and massive-scale mRNA sequencing, a flow cytometric approach was developed to isolate P. xanthii haustoria free of visible contaminants. Next, several commercial kits were used to isolate total RNA and to construct the cDNA and Illumina libraries that were finally sequenced by the Illumina NextSeq system. Using this approach, the maximum amount of information from low-quality RNA that could be obtained was used to accomplish the de novo assembly of the P. xanthii haustorial transcriptome. The subsequent analysis of this transcriptome and comparison with the epiphytic transcriptome allowed us to identify the importance of several biological processes for haustorial cells such as protection against reactive oxygen species, the acquisition of different nutrients and genetic regulation mediated by non-coding RNAs. In addition, we could also identify several secreted proteins expressed exclusively in haustoria such as cell adhesion proteins that have not been related to powdery mildew biology to date. Conclusions This work provides a novel approach to study the molecular aspects of powdery mildew haustoria. In addition, the results of this study have also allowed us to identify certain previously unknown processes and proteins involved in the biology of powdery mildews that could be essential for their biotrophy and pathogenesis. Electronic supplementary material The online version of this article (10.1186/s12864-019-5938-0) contains supplementary material, which is available to authorized users.

A haustorial‐expressed lytic polysaccharide monooxygenase from the cucurbit powdery mildew pathogen Podosphaera xanthii contributes to the suppression of chitin‐triggered immunity

“…The high expression of PxLPMO1 , which was the 13th most highly expressed gene in the haustorium and the most highly expressed gene among the genes encoding haustorium‐specific secreted proteins (Polonio, Seoane, et al., 2019), suggested that it plays an important role in P. xanthii biology. To validate the role of PxLPMO1 , the Agrobacterium tumefaciens ‐mediated host‐induced gene silencing (ATM‐HIGS) assay was used.…”

“…xanthii (Polonio, Seoane, et al, 2019) allowed us to identify an effector candidate, PHEC27213, that had typical effector features, such as no annotated function, a small size, and a wave-like expression pattern (Hacquard, 2014;Pedersen et al, 2012). Furthermore, PHEC27213 was the most highly expressed, haustorium-specific, putative secreted protein (Polonio, Seoane, et al, 2019), pointing to its key role in haustorial physiology. In this work, we performed a molecular characterization of PHEC27213 to elucidate its putative function as a chitin manipulation-related protein.…”

“…(b) Panoramic view of the PxLPMO1 protein with the DP7 molecule docked in the proposed binding site. DP7 binds to PxLPMO1 via residues 191,190,189,and 118 (yellow), which allow the interaction of the chitin heptamer with residues 1 and 52 of the histidine brace active site (green) gene among the genes encoding haustorium-specific secreted proteins (Polonio, Seoane, et al, 2019) (e) Effect of the infiltration of the products from the PxLPMO1 (PHEC27213) enzymatic reaction on the oxidative burst. The supernatants from the reaction products described in Figure 3a were infiltrated into melon cotyledons, and then the samples were processed for detection of H 2 O 2 by the DAB uptake method.…”

“…For gene expression analysis, total RNA was extracted and used to synthetize cDNA as described above. As normalization reference genes, the P. xanthii translation elongation factor 1-alpha gene PxEF1 (MK249653) and the C. melo actin-7 gene CmACT7 (XM_008462689.2) were used (Polonio, Pineda, et al, 2019;Polonio, Seoane, et al, 2019). For the molecular estimation of fungal biomass, total DNA was isolated from agroinfiltrated and infected melon cotyledons as described above.…”

“…The first evidence in this regard has been the recent identification of effectors with chitinase activity (EWCAs), a family of conserved chitinases, secreted mainly by hyphae, that suppress chitin signalling by catalysing immunogenic chitooligosaccharides (Martínez-Cruz et al, 2021). In the haustorial transcriptome of P. xanthii, a unigene encoding a small secreted protein without an annotated function but predicted by protein modelling to be a putative lytic polysaccharide monooxygenase (LPMO) with a chitin-binding domain, was found among the top 50 expressed genes; it was the most highly expressed gene among those encoding proteins specifically expressed in the haustorium (Polonio, Seoane, et al, 2019). LPMOs are a class of recently characterized enzymes that are able to oxidize different recalcitrant polysaccharides, including chitin (Hemsworth et al, 2015;Vaaje-Kolstad et al, 2010).…”