Towards identifying pathogenic determinants of the chickpea pathogen Ascochyta rabiei

Abstract

Ascochyta blight is a serious disease of cool-season grain legumes (chickpea, faba bean, lentil and pea) caused by fungal species of the anamorphic genus Ascochyta and related genera. Despite extensive studies on the biology, ecology, epidemiology and management of the disease, little is known about the pathogenic determinants of these pathogens. This research aims at using Ascochyta rabiei as a model for the genus in investigating genetic factors of pathogenicity, with the ultimate goal of elucidating pathogenic mechanisms. Three advances were made: (1) insertional mutants with altered pathogenicity were identified through in vivo screening, and genomic regions adjacent to the insertion sites in selected mutants were determined; (2) a phage library of A. rabiei genomic DNA was constructed, and the library was estimated to provide complete coverage of the A. rabiei genome. This library was used successfully to recover clones with DNA adjacent to insertional mutation sites and to isolate specific genes; (3) DNA probes specific for an acyl-CoA ligase (cps1) and a polyketide synthase gene (pks1) were developed and library clones containing the corresponding genomic regions were identified from the phage library. These advances provide the foundation and necessary tools for experimentation of ectopic complementation assays and targeted mutagenesis to elucidate the genetic mechanisms of pathogenicity of A. rabiei.

Agrobacterium-mediated transformation of Guignardia citricarpa: An efficient tool to gene transfer and random mutagenesis

“…The ATMT method is very attractive to obtain phenotypes mutants and identify genes related to these via random insertional mutagenesis. Responsive gene related to the pathogenic character of fungi species, could be identified or knocked out by this technique, allowing the study of several pathogenic systems (Rho et al 2001;Combier et al 2003;Leclerque et al 2004;Rogers et al 2004;Walton et al 2005;White & Chen 2007;Zhong et al 2007;Talhinhas et al 2008). The establishment of this methodology for G. citricarpa future experiments can be efficiently performed in order to achieve similar results.…”

In planta Identification of Putative Pathogenicity Factors from the Chickpea Pathogen Ascochyta rabiei by De novo Transcriptome Sequencing Using RNA-Seq and Massive Analysis of cDNA Ends

“…A total of 59 transcripts with homology to genes included in the PHI database and known to be involved in pathogenicity in other pathogenic fungi were more expressed during infection (Additional file 6 ). The transcripts corresponding to two A. rabiei genes previously identified in response to oxidative stress ( Ar125, Ar126 ) and the polyketide synthase pks2 were also found to be over-expressed during infection, but not pks1 and cps1 (Tenhaken et al, 1997 ; White and Chen, 2007 ; Akamatsu et al, 2010 ; Singh et al, 2012 ).…”

“…Using the BLASTN algorithm we additionally identified the sequences corresponding to some already known A. rabiei genes in our transcriptome, and examined whether they were up-regulated during infection. These genes encoded a cutinase (Tenhaken et al, 1997 ), the polyketide synthases pks1 and pks2 , the putative A. rabiei virulence factor cps1 (White and Chen, 2007 ; Akamatsu et al, 2010 ), or were induced after oxidative stress (Singh et al, 2012 ).…”

Transformação genética e patogenicidade de Guignardia citricarpa

“…Agrobacterium permite a obtenção de um maior número de transformantes (AMEY et al, 2002;CAMPOY et al, 2003;FITZGERALD et al, 2003;RODRÍGUEZ-TOVAR et al, 2005 GROOT et al, 1998;COVERT et al, 2001, MORIOKA et al, 2006. Essas características tornam o método de agrotransformação bastante atraente quando se deseja obter fenótipos mutantes e identificar os genes a estes relacionados via mutagênese insercional aleatória (RHO et al, 2001;COMBIER et al, 2003;ROGERS et al, 2004;LECLERQUE et al, 2004;WALTON et al, 2005;WHITE;CHEN, 2007;ZHONG et al, 2007;TALHINHAS et al, 2008). Outra utilidade atrativa é quando se deseja realizar estudos para mutagênese direcionada ou em experimentos de nocaute gênico (GOUKA et al, 1999;ZWIERS et al, 2001;ZHANG et al, 2003;ZEILINGER, 2004;SUGUI et al, 2005;KHANG et al, 2005;GONG et al, 2007;GODIO;MOON et al, 2008;MARTÍN, 2009).…”

“…(MICHIELSE et al, 2005;WALTON;HEITMAN, 2005;JEON et al, 2007;BLAISE et al, 2007;HUSER et al, 2009). (RHO et al, 2001;COMBIER et al, 2003;ROGERS et al, 2004;LECLERQUE et al, 2004;WALTON et al, 2005;WHITE;CHEN, 2007;ZHONG et al, 2007;TALHINHAS et al, 2008). Com o estabelecimento da metodologia de agrotransformação para G. citricarpa, futuros experimentos para esse fim podem ser realizados de forma eficiente.…”