Project description:The late blight pathogen, Phytophthora infestans has a broad host range within the Solanaceae family, including yellow potato (Solanum phureja). The disease caused by P. infestans in S. phureja is poorly understood and is a major concern in Colombia. Expressed Sequence Tag (EST) libraries obtained from a normalized library constructed from healthy plant tissue revealed high levels of sequence similarity between S. phureja and S. tuberosum. Then, utilizing Serial Analysis of Gene Expression and high-throughput sequencing (SAGE-Solexa), we characterized yellow potato gene expression during infection by P. infestans. Four-week-old yellow potato plants were inoculated with P. infestans and were collected at 12 and 72 hours post inoculation for RNA extraction. We detected differentially expressed genes by comparing inoculated to non-inoculated and resistant to susceptible plants. The discovery and characterization of the proteins mediating this host–pathogen interaction enable the understanding of the pathosystem and is the key for developing resistant plants. Keywords: SAGE-Solexa, inoculation response, transcript profiling, Solanum phureja, Phytophthora infestans
Project description:The late blight pathogen, Phytophthora infestans has a broad host range within the Solanaceae family, including yellow potato (Solanum phureja). The disease caused by P. infestans in S. phureja is poorly understood and is a major concern in Colombia. Expressed Sequence Tag (EST) libraries obtained from a normalized library constructed from healthy plant tissue revealed high levels of sequence similarity between S. phureja and S. tuberosum. Then, utilizing Serial Analysis of Gene Expression and high-throughput sequencing (SAGE-Solexa), we characterized yellow potato gene expression during infection by P. infestans. Four-week-old yellow potato plants were inoculated with P. infestans and were collected at 12 and 72 hours post inoculation for RNA extraction. We detected differentially expressed genes by comparing inoculated to non-inoculated and resistant to susceptible plants. The discovery and characterization of the proteins mediating this host–pathogen interaction enable the understanding of the pathosystem and is the key for developing resistant plants. Keywords: SAGE-Solexa, inoculation response, transcript profiling, Solanum phureja, Phytophthora infestans Four-week-old yellow potato (Solanum phureja) plants were inoculated with Phytophthora infestans and were collected and flash frozen in liquid nitrogen at 12 and 72 hours post inoculation, as well as mock inoculated, for RNA extraction. 2 yellow potato cultivars (resistant and susceptible) were used for each experiment. Mock inoculated plants were collected in each replicate. RNA obtained from each of the three biological replicates was pooled to obtain a single RNA sample for each timepoint X cultivar combination. A total of 6 different SAGE libraries were thus obtained. For all libraries, Illumina sequencing was performed at Canada´s Michael Smith Genome Sciences Centre.
Project description:Background: The oomycete Phytophthora infestans possesses active RNA silencing pathways, which presumably enable this plant pathogen to control the large numbers of transposable elements present in its 240 Mb genome. Small RNAs (sRNAs), central molecules in RNA silencing, are known to also play key roles in this organism, notably in regulation of critical effector genes needed for infection of its potato host. Results: To identify additional classes of sRNAs in oomycetes, we mapped deep sequencing reads to transfer RNAs (tRNAs) thereby revealing the presence of 19-40 nt tRNA-derived RNA fragments (tRFs). Northern blot analysis identified abundant tRFs corresponding to half tRNA molecules. Some tRFs accumulated differentially during infection, as seen by examining sRNAs sequenced from P. infestans-potato interaction libraries. The putative connection between tRF biogenesis and the canonical RNA silencing pathways was investigated by employing hairpin RNA-mediated RNAi to silence the genes encoding P. infestans Argonaute (PiAgo) and Dicer (PiDcl) endoribonucleases. By sRNA sequencing we show that tRF accumulation is PiDcl1-independent, while Northern hybridizations detected reduced levels of specific tRNA-derived species in the PiAgo1 knockdown line. Conclusions: Our findings extend the sRNA diversity in oomycetes to include fragments derived from non-protein-coding RNA transcripts and identify tRFs with elevated levels during infection of potato by P. infestans. Small RNA sequence data from Phytophthora infestans-infected potato leaf tissue and P. infestans mycelium tissue. Three infection stage time-points. Two P. infestans lines: 88089 (wild-type) and PiDcl1 (transformant PiDcl1 knock-down). No replicates. Total number of samples: 8.
Project description:Potato (Solanum tuberosum) late blight disease caused by Phytophthora. infestans is one of the most critical crop diseases in the world. We investigated the proteome of Solanum tubersoum (cv. Sarpo Mira) by quantitative proteomics after foliar application of encysted zoospore suspension from P. infestans isolate. Also, we examined changes in protein abundance of cv. Sarpo Mira in response to P. infestans infection at three key timepoints 0 hpi (L), 48 hpi (M) and 120 hpi (H), divided into early and late disease stages and analyzed the protein-protein interaction during the disease stages. In response to P. infestans oomycete infection, the iTRAQ comparative proteome survey revealed a total of 1229 differentially expressed proteins (DEPs) in the pairwise comparison of disease stages, which included DEPs specific to early disease stage, DEPs specific to late disease stage and commonly shared DEPs. Over 80% of the change in protein abundance were up-regulated in the early stages of infection while about 61% of the DEPs in the later disease stage were down-regulated. The analysis of expression patterns, functional category, and enrichment tests highlighted significant coordination and enrichment of cell wall-associated defense response proteins in the early stages and cellular protein modification process as well as membrane protein complex formation in the late stages. Furthermore, strong protein interactions were observed in the late disease stage that might relate to disease pathogenesis.