Discovery of Phytophthora cinnamomi RxLR effector genes expressed in avocado during infection

Abstract

Phytophthora cinnamomi is an oomycete that targets a broad range of plants, including several economically important forestry and agricultural crops. It is the causal agent of Phytophthora Root Rot, and causes significant economic losses within the agricultural and forestry industries. Recently, the use of effector molecules by pathogenic oomycetes during plant infection has become a subject of great interest to researchers. One class of these molecules, the RxLR effectors, has become a focus of Phytophthora research, and hundreds of putative RxLR effector genes have been predicted by bioinformatic analysis of Phytophthora genomic sequences. The characterization and validation of these effectors remains an ongoing process. This study identified several P. cinnamomi RxLR genes upregulated during infection of a susceptible avocado rootstock. The genes were subjected to in silico analysis of expected RxLR characteristics and prediction of coding regions from the genomic sequence. Predictions were then validated by analysis of DNA sequences and the use of RNA-seq data, which were used to manually annotate these effector genes. The final prediction of RxLR proteins was then compared to the sequences of validated RxLRs in other Phytophthora species to enable inference of possible functions of the annotated genes. In this study, a total of 25 P. cinnamomi candidate RxLRs were identified, which were proposed to play a role during avocado infection. While expression of this number of candidate RxLRs is relatively small, these candidates may represent effectors which are expressed specifically in this host-pathogen interaction, or may be a set of “elite” effectors which contribute to virulence in all hosts. The candidate genes were analysed for the presence of the desired motifs, and a subset of 16 RxLRs were chosen for further analysis. The expression profiles of these genes were investigated further, and it was found that four of the candidates were expressed most highly at 24 hpi, which correlates with expression profiles of RxLRs in other species. Twelve of the candidate RxLRs had expression profiles which were not similar to those which have been demonstrated for other RxLRs, while four were not significantly upregulated during specific timepoints of infection. These results warrant further investigation to determine the relevance of these unique expression profiles, which may present new insights into expression patterns of RxLR effectors. Several of the 16 candidate effector genes were present in multiple copies in the P. cinnamomi genome, providing evidence for their roles in plant infection. Transcriptome data was used to manually annotate the genes, and the resulting protein predictions for most of the candidates were different from those originally predicted by gene prediction software. Not one of the prediction software used in this experiment accurately predicted the coding regions for all the genes – providing a substantial argument for the need for manual annotation of candidate effectors. Phylogenetic analysis allowed functional inferences to be made for five of the candidate effectors, based on their shared evolutionary history with RxLRs characterized in other Phytophthora species. While no functional assays have been carried out for these candidate effectors yet, their identification as putative RxLRs presents a starting point for further investigation into their functions in planta. This study presents the first report of P. cinnamomi RxLRs with confirmed sequences and expression profiles, and as such offers the first insights into infection of avocado by this pathogen at the molecular level.