Identification and characterization of polygalacturonases in Phytophthora cinnamomi

Abstract

Phytophthora cinnamomi is an economically important plant pathogen and has caused devastating losses to the avocado industry. For the pathogen to penetrate the host plant, it secretes a variety of cell wall degrading enzymes (CWDEs). Polygalacturonases (PGs) are the first CWDEs secreted by the pathogen during infection and have been reported to play an important role in pathogenesis. Although a large PG gene family in P. cinnamomi has been reported, knowledge on the in-depth bioinformatic analysis and characterization of these genes is lacking. Furthermore, the role of PGs from P. cinnamomi during avocado infection is not fully understood. In this study, PGs were computationally identified using the genome sequence of P. cinnamomi. A total of 26 PG sequences were identified, with 13 PG sequences containing all the characteristics of a full-length PG protein. We further investigated the evolution of the 13 full-length PG genes on a genome-wide scale. The proteins sequences encoded by these genes clustered into two distinct clades based on their putative N-linked glycosylation sites. Furthermore, intron evolution was analyzed in these genes, which revealed intron gain and loss events, suggesting that introns in the PG genes from P. cinnamomi follow a systemic combined theory of “intron early” and “intron late”. Putative function was inferred on the full-length P. cinnamomi PG (pcpg) genes based on sequence similarity to PGs from other oomycete species. Furthermore, two separate phylogenetic trees of oomycetes and fungal species were constructed to investigate clustering of the PGs based on trophic lifestyle, revealing that PGs do not cluster based on the trophic lifestyle. To further investigate the PG genes from P. cinnamomi, a time-course differential gene expression analysis was conducted, and candidate pathogenicity genes were identified. We further compared the expression of pcpg genes during infection of the susceptible R0.12 versus the partially resistance Dusa® rootstock. Predominant pcpg gene expression was observed in the Dusa® rootstock when compared to R0.12, suggesting the need for the pathogen to secrete more PGs to evade the defense responses signaled by the partially resistant rootstock, allowing the pathogen to establish infection.

This study provides a comprehensive understanding of PG genes in P. cinnamomi and highlights candidate PG pathogenicity genes from P. cinnamomi important for infection of avocado. It also presents the first report on the evolution of introns in PGs of P. cinnamomi. Furthermore, the study provides a framework for functional characterization of the putative pathogenicity genes, providing knowledge that will assist in targeted inhibition of these genes, thereby reducing the virulence of the pathogen.