Prediction of the in planta Phakopsora pachyrhizi secretome and potential effector families

Abstract

Asian soybean rust (ASR), caused by the obligate biotrophic fungus Phakopsora pachyrhizi, can cause losses greater than 80%. Despite its economic importance, there is no soybean cultivar with durable ASR resistance. In addition, the P. pachyrhizi genome is not yet available. However, the availability of other rust genomes, as well as the development of sample enrichment strategies and bioinformatics tools, has improved our knowledge of the ASR secretome and its potential effectors. In this context, we used a combination of laser capture microdissection (LCM), RNAseq and a bioinformatics pipeline to identify a total of 36 350 P. pachyrhizi contigs expressed in planta and a predicted secretome of 851 proteins. Some of the predicted secreted proteins had characteristics of candidate effectors: small size, cysteine rich, do not contain PFAM domains (except those associated with pathogenicity) and strongly expressed in planta. A comparative analysis of the predicted secreted proteins present in Pucciniales species identified new members of soybean rust and new Pucciniales- or P. pachyrhizi-specific families (tribes). Members of some families were strongly up-regulated during early infection, starting with initial infection through haustorium formation. Effector candidates selected from two of these families were able to suppress immunity in transient assays, and were localized in the plant cytoplasm and nuclei. These experiments support our bioinformatics predictions and show that these families contain members that have functions consistent with P. pachyrhizi effectors.

New insights into Phakopsora pachyrhizi infection based on transcriptome analysis in planta

“…When 58 transcripts were compared to the secreted sequences identified by Link et al (2014) in the haustorium transcriptome of P. pachyrhizi , it was possible to identify 44 sequences similar to 12 of the transcripts that were predicted as secreted. In addition, three transcripts that were found among the top expressed transcripts common to both genotypes (de_novo_2238, de_novo_5381, and de_novo_5849) were also functionally validated by Carvalho et al (2016) .…”

“…Each cap was used for pulses ranging from 1500–2000 corresponding to the collection of approximately the same number of cells. Samples were collected for both resistant and susceptible plants ( Carvalho et al , 2016 ).…”

“…Since the nuclear genome of P. pachyrhizi has not yet been sequenced, most of the molecular information about this pathogen has been obtained from work involving transcriptome sequencing associated with bioinformatics analyses. Thus, a moderate repertoire of candidate gene sequences expressed in germinated urediniospores, appressorium, haustorium, and uredinia have become available ( Posada-Buitrago and Frederick, 2005 ; Tremblay et al , 2009 , 2012 , 2013 ; Stone et al , 2012 ; Link et al , 2014 ; Carvalho et al , 2016 ). Most of these studies focused on the characterization of specific molecular mechanisms during the infection process, with a concentration on analyses of specific structures of the pathogen, such as the urediniospores and haustorium, or of the total infected leaf.…”

Advances in understanding obligate biotrophy in rust fungi

“…For instance, six candidates showed suppression of plant cell death triggered by Pseudomonas syringae pv. tomato DC3000 and an accumulation in nucleus and cytosol after transient expression in N. benthamiana (de Carvalho et al ., ). In another study, two out of six selected candidates were found to increase P. infestans infection in N. benthamiana , suggesting a role in the virulence of the soybean rust fungus (Kunjeti et al ., ).…”

Identification of Plasmodiophora brassicae effectors — A challenging goal

“…In C. fulvum , cys-rich effectors can inhibit and protect against plant hydrolytic enzymes, such as proteases, glucanases, and chitinases [32]. Cys-rich small-secreted proteins have also been identified as major effectors in the obligate biotrophic pathogens Melampsora larici-populina [35], and the Asian soybean rust fungus Phakopsora pachyrhizi [42], where one of the cys-rich small proteins identified as an effector has been shown to suppress plant immunity [43]. …”