Identification Of Small RNAS Associated With Development And Virulence In Phytophthora Sojae

Pathogens cause severe damages to the survival, growth and reproduction of plant. The pathogenic oomycetes are destructive to crop production and threaten natural ecosystems. Oomycete are diploid eukaryotic microorganisms, which are similar to filamentous fungi in morphology but close to brown algae and diatoms in evolution, making fungicides ineffective against oomycetes. Well-known oomycetes include Phytophthora, Peronospora, Pythium and Albugo. The causal agent of Irish potato famine, Phytophthora infestans, causes $6.7 billion potato losses annually. Phytophthora sojae causes damping-off of seedlings and root rot of soybean, with an annual cost of $1-2 billion worldwide.Use of host resistance is the most effective approach for crop disease control. However, genetic variation of pathogens, including possible epigenetic variations that were observed in the genetic variation of Phytophthora, causes frequent crop resistance failure. Small RNAs, such as si RNA, mi RNA and pi RNA, are able to pair complementarily with its antisense transcript locally and lead to transcriptional and posttranscriptional gene silencing. They are important molecules in epigenetical regulation but little is known on diversity, characteristics, functions and underlying regulatory mechanisms of Phytophthora small RNAs. Therefore, we use P. sojae as a model to study the small RNA involved in the pathogen development and virulence variation, and the main findings are as follows:1. By using deep sequencing, computational identification, conventional cloning and Northern hybridization, we identified a large amount of ts RNAs(t RNA-derived small RNA) encoded by the P. sojae genome. Using algorithm developed for ts RNA identification and annotation, we further analyzed the small RNA re-sequencing data for the vegetative hyphae stage of P. sojae. The results are consistent with Northern blotting, being the t RNA reads enriched in 29-36 nt and peaked at 33 nt. The first nucleotide of t RNA reads is predominately G, followed by T. In the dataset within 28- 45 nt, the t RNA reads constitute to 12.34 % of the total reads, while the most abundant ts RNA-Gly CCC-5p comprises 2.05 % of the total reads.2. Genome analysis and Northern hybridization showed that ts RNA is a class of conserved small RNAs. Northern analysis also showed that the expression patterns of ts RNA are relatively conserved, being highly accumulated in oospores, mycelia and sporangia, and much lower in the infection stage and germinated cysts, while nearly not detected in cysts. Bioinformatics analyses showed a large set of potential ts RNA target candidates. Digital gene expression profiling and real-time quantitative RT-PCR results indicated that the expression levels of the most target genes are negatively correlated with accumulation levels ts RNAs, suggesting that ts RNAs are functional and may down-regulate gene expression in P. sojae.3. By using RNA quantification and RLM-RACE technologies(RNA ligation mediated-rapid amplification of c DNA ends), we confirmed degradation of five target genes in P. sojae mycelia. However, the degradation of target transcripts was not in the ts RNA binding sites. This is distinct from mi RNA-mediated degradation of target transcripts. Consistent with the RLM-RACE experiments, high-throughput PARE(parallel analysis of RNA ends) analysis showed that ts RNA-mediated degradation of target transcripts occurs near but not at the binding sites, being slightly in favor of the downstream region. Transient transformation analysis in P. sojae demonstrated that ts RNAs repress expression of artificial GUS targets, in a sequence-specific manner.4. By deep sequencing analysis of the effector gene Avr1b-1-silencing strain and Northern hybridization, two types of small RNAs homologous to Avr1b-1 were identified, the potential silencing trigger Avr1b-1-s RNA and the signal molecules Avr1b-1-si RNAs. Hybridization results also indicated that both types of small RNAs accumulated in the virulent but not in the avirulent strain. In addition, the trigger Avr1b-1-s RNA accumulated 12-24 hours earlier than the silencing signal Avr1b-1-si RNAs. Further bioinformatics analyses and Northern hybridization showed that vast small RNAs are associated with RXLR effector genes, and their expression levels were negatively correlated.5. Genome analysis and RT-PCR analysis indicated bidirectional transcription of Avr1b-1 and the natural antisense transcripts being the precursor of Avr1b-1-s RNA, the final product Avr1b-1-s RNA may regulates the expression level of Avr1b-1. The RT-PCR data also suggested that the differentially expressed Avr1b-1(-) is responsible for the differentially accumulated Avr1b-1-s RNA trigger in the virulent and avirulent strains. Further sequence analysis showed that the expression levels of Avr1b-1(+) and Avr1b-1(-) are likely associated with the presence or absence of the ~10 nt INDELs(insertions and deletions) in their promoter regions.