EST Mining and Functional Expression Assays Identify Extracellular Effector Proteins From the Plant PathogenPhytophthora

Abstract

Plant pathogenic microbes have the remarkable ability to manipulate biochemical, physiological, and morphological processes in their host plants. These manipulations are achieved through a diverse array of effector molecules that can either promote infection or trigger defense responses. We describe a general functional genomics approach aimed at identifying extracellular effector proteins from plant pathogenic microorganisms by combining data mining of expressed sequence tags (ESTs) with virus-based high-throughput functional expression assays in plants. PexFinder, an algorithm for automated identification of extracellular proteins from EST data sets, was developed and applied to 2147 ESTs from the oomycete plant pathogen Phytophthora infestans. The program identified 261 ESTs (12.2%) corresponding to a set of 142 nonredundant Pex (Phytophthora extracellular protein) cDNAs. Of these, 78 (55%) Pex cDNAs were novel with no significant matches in public databases. Validation of PexFinder was performed using proteomic analysis of secreted protein of P. infestans. To identify which of the Pex cDNAs encode effector proteins that manipulate plant processes, high-throughput functional expression assays in plants were performed on 63 of the identified cDNAs using an Agrobacterium tumefaciens binary vector carrying the potato virus X (PVX) genome. This led to the discovery of two novel necrosis-inducing cDNAs, crn1 and crn2, encoding extracellular proteins that belong to a large and complex protein family in Phytophthora. Further characterization of the crn genes indicated that they are both expressed in P. infestans during colonization of the host plant tomato and that crn2 induced defense-response genes in tomato. Our results indicate that combining data mining using PexFinder with PVX-based functional assays can facilitate the discovery of novel pathogen effector proteins. In principle, this strategy can be applied to a variety of eukaryotic plant pathogens, including oomycetes, fungi, and nematodes.

Agroinfiltration and PVX Agroinfection in Potato and <em>Nicotiana benthamiana</em>

“…Normally, expanding cell death can be found at the sites about two weeks after tooth-pick inoculation. As shown in both figure panels, expanding cell death is present at the sites that were tooth-pick inoculated with pGR106-CRN2 (positive control using the effector crinkler Crn2), which is a general cell death inducing gene from P. infestans 27 . Apart from minor response to wounding, no expanding cell death is noted at the sites that were tooth-pick inoculated with the pGR106-empty (negative control).…”

A novel non-protein-coding infection-specific gene family is clustered throughout the genome of Phytophthora infestans

“…ORFs encoding putative RXLR sequences Kamoun 2006;Rehmany et al, 2005) were identified using the combined procedures outlined by Bhattacharjee et al (2006) and Torto et al (2003). A total of 159 ORFs putatively encoding secreted RXLR proteins were identified on the 135 supercontig sequences containing matches to Pinci1.…”

RXLR-Mediated Entry of Phytophthora sojae Effector Avr1b into Soybean Cells Does Not Require Pathogen-Encoded Machinery

“…Since each of these avirulence proteins appears to enter the host cells, they have been inferred to be effector proteins. Several fungal and oomycete effector proteins that are not associated with resistance gene interactions have also been shown or inferred to enter plant cells, including Phytophthora elicitin (Tyler, 2002) and crinkler (Torto et al, 2003) proteins, the RTP1p protein of the bean rust Uromyces fabae (Kemen et al, 2005), and the ToxA protein toxin of Pyrenophora tritici-repentis (Manning and Ciuffetti, 2005). In all cases, the mechanisms by which these proteins might enter the cell are unknown.…”