Transcription profiling and identification of infection-related genes in Phytophthora cactorum.

Phytophthora cactorum, an oomycete pathogen, infects more than 200 plant species within several plant families. To gain insight into the repertoire of the infection-related genes of P. cactorum, Illumina RNA-Seq was used to perform a global transcriptome analysis of three life cycle stages of the pathogen, mycelia (MY), zoospores (ZO) and germinating cysts with germ tubes (GC). From over 9.8 million Illumina reads for each library, 18,402, 18,569 and 19,443 distinct genes were identified for MY, ZO and GC libraries, respectively. Furthermore, the transcriptome difference among MY, ZO and GC stages was investigated. Gene ontology (GO) and KEGG pathway enrichment analyses revealed diverse biological functions and processes. Comparative analysis identified a large number of genes that are associated with specific stages and pathogenicity, including 166 effector genes. Of them, most of RXLR and NLP genes showed induction while the majority of CRN genes were down-regulated in GC, the important pre-infection stage, compared to either MY or ZO. And 14 genes encoding small cysteine-rich (SCR) secretory proteins showed differential expression during the developmental stages and in planta. Ectopic expression in the Solanaceae indicated that SCR113 and one elicitin PcINF1 can trigger cell death on Nicotiana benthamiana, tobacco (N. tabacum) and tomato (Solanum lycopersicum) leaves. Neither conserved domain nor homologues of SCR113 in other organisms can be identified. Collectively, our study provides a comprehensive examination of gene expression across three P. cactorum developmental stages and describes pathogenicity-related genes, all of which will help elucidate the pathogenicity mechanism of this destructive pathogen.

Transcriptomic analysis of the phytopathogenic oomycete Phytophthora cactorum provides insights into infection-related effectors.

BACKGROUND: Phytophthora cactorum, a hemibiotrophic oomycete pathogen, can cause destructive diseases on numerous crops worldwide, leading to essential economic losses every year. However, little has been known about its molecular pathogenicity mechanisms. To gain insight into its repertoire of effectors, the P. cactorum transcriptome was investigated using Illumina RNA-seq. RESULTS: We first demonstrated an in vitro inoculation method that can be used to mimic natural cyst germination on host plants. Over 28 million cDNA reads were obtained for five life cycle stages (mycelium, sporangium, zoospore, cyst and germinating cyst) and de novo assembled into 21,662 unique genes. By comparisons with 11 public databases, 88.99% of the unique genes were annotated, including 15,845 mapped to the gene models of the annotated relative Phytophthora infestans. Using TribeMCL, 5,538 gene families conserved across P. cactorum and other three completely sequenced Phytophthora pathogen species were determined. In silico analyses revealed that 620 P. cactorum effector homologues including 94 RXLR effector candidates matched known or putative virulence genes in other oomycetes. About half of the RXLR effector candidates were predicted to share a conserved structure unit, termed the WY-domain fold. A subset of the effector genes were checked and validated by PCR amplification. Transcriptional experiments indicated that effector genes were differentially expressed during the life cycle and host infection stages of P. cactorum. Ectopic expression in Nicotiana benthamiana revealed that RXLR, elicitin and NLP effectors can trigger plant cell death. These effectors are highly conserved across oomycete species. Single nucleotide polymorphisms for RXLR effectors were detected in a collection of P. cactorum isolates from different countries and hosts. CONCLUSIONS: This study demonstrates the comprehensive sequencing, de novo assembly, and analyses of the transcriptome of P. cactorum life cycle stages. In the absence of genome sequence, transcriptome data is important for infection-related gene discovery in P. cactorum, as demonstrated here for the effector genes. The first look at the transcriptome and effector arsenal of P. cactorum provides valuable data to elucidate the pathogenicity basis of this broad-host-range pathogen.