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Transformation by growth onto agro-infiltrated tissues (TGAT), a simple and efficient alternative for transient transformation of the cucurbit powdery mildew pathogen Podosphaera xanthii.

ABSTRACT: A major limitation of molecular studies in powdery mildew fungi (Erysiphales) is their genetic intractability. This is because they are obligate biotrophs. In these parasites, biotrophy is determined by the presence of haustoria, which are specialized structures of parasitism that play an essential role in the acquisition of nutrients and the deliverance of effectors. Podosphaera xanthii is the main causal agent of cucurbit powdery mildew and a major limitation for crop productivity. In a previous study using P. xanthii conidia, we showed, for the first time, the transformation of powdery mildew fungi by Agrobacterium tumefaciens. In this work, we hypothesized that the haustorium could also act as a natural route for the acquisition of DNA. To test our hypothesis, melon cotyledons were agro-infiltrated with A. tumefaciens that contained diverse transfer DNA (T-DNA) constructs harbouring different marker genes under the control of fungal promoters and, after elimination of the bacterium, the cotyledons were subsequently inoculated with P. xanthii conidia. Our results conclusively demonstrated the transfer of different T-DNAs from A. tumefaciens to P. xanthii, including two fungicide resistance markers (hph and tub2), a reporter gene (gfp) and a translational fusion (cfp-PxEC2). These results were further supported by the co-localization of translational fluorescent fusions of A. tumefaciens VirD2 and P. xanthii Rab5 proteins into small vesicles of haustorial and hyphal cells, suggesting endocytosis as the mechanism for T-DNA uptake, presumably by the haustorium. From our perspective, transformation by growth onto agro-infiltrated tissues (TGAT) is the easiest and most reliable method for the transient transformation of powdery mildew fungi.

SUBMITTER: Martinez-Cruz J

PROVIDER: S-EPMC6638186 | biostudies-literature | 2018 Nov

REPOSITORIES: biostudies-literature

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Transformation by growth onto agro-infiltrated tissues (TGAT), a simple and efficient alternative for transient transformation of the cucurbit powdery mildew pathogen Podosphaera xanthii.

Martínez-Cruz Jesús J Romero Diego D De Vicente Antonio A Pérez-García Alejandro A

Molecular plant pathology 20181010 11

A major limitation of molecular studies in powdery mildew fungi (Erysiphales) is their genetic intractability. This is because they are obligate biotrophs. In these parasites, biotrophy is determined by the presence of haustoria, which are specialized structures of parasitism that play an essential role in the acquisition of nutrients and the deliverance of effectors. Podosphaera xanthii is the main causal agent of cucurbit powdery mildew and a major limitation for crop productivity. In a previo ...[more]

PMID: 30073764

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Project description:BackgroundPodosphaera 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.ResultsPrior 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.ConclusionsThis 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.

Project description:Powdery mildew is one of the most destructive diseases and the major cause to the production losses of cucurbit worldwide. A number of strategies have been developed and applied to discover suitable and safer methods to manage the occurrence of powdery mildew disease in pumpkins (Cucurbita pepo L.), but information is limited in screening tolerant germplasms and exploring their mechanisms in preventing the disease occurrence at physiological, biochemical, and molecular levels. Therefore, we investigated the responses of two commercial pumpkin cultivars to Podosphaera xanthii infection. Compared with mock-inoculated seedlings, few small and sparse powdery areas were observed on the leaves of the Sixing F1 cultivar on the 13th day after inoculation with P. xanthii, whereas a large number of diseased powdery areas and a layer of white powdery mildew were observed on the surface of Jin12 F1 leaves. The inoculation duration (7, 9, 11, and 13 days) significantly and continuously increased the disease incidence and index of pumpkin seedlings. The contents of H2O2, MDA, lignin, and total phenolics in the leaves of Sixing F1 and Jin12 F1 cultivars were markedly increased after inoculation with P. xanthii. However, the Sixing F1 cultivar exhibited much less reactive oxygen species (ROS) accumulation, a lower rate of lipid peroxidation, and a higher level of lignin and total phenolics contents after inoculation than the Jin12 F1 cultivar. Compared with untreated control pumpkin seedlings, significantly higher activities and gene expressions of the phenylpropanoids pathway enzymes (PAL and PPO), ROS scavenging defense enzymes (SOD, CAT, POD, and APX), and other salicylic acid (SA) signaling pathway marker genes were observed in the leaves of both cultivars after P. xanthii inoculation at different inoculation time points. These enhancements were significantly higher in Sixing F1 than Jin12 F1. Our results indicate that the Sixing F1 cultivar exhibited a much stronger ability in resistance to P. xanthii infection than the Jin12 F1 cultivar. Our results suggest that one possible mechanism of C. pepo cultivars to prevent the pathogen P. xanthii infection is by activating and enhancing the activity and gene expression of the phenylpropanoids pathway to synthesize phenolic substances and lignin, ROS scavenging defense enzymes to eliminate the harmful effects of ROS, and signaling pathway marker gene expression to improve plant disease resistance.

Dataset Information

Transformation by growth onto agro-infiltrated tissues (TGAT), a simple and efficient alternative for transient transformation of the cucurbit powdery mildew pathogen Podosphaera xanthii.

Publications

Transformation by growth onto agro-infiltrated tissues (TGAT), a simple and efficient alternative for transient transformation of the cucurbit powdery mildew pathogen Podosphaera xanthii.

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