| Grape, as one of the most economically valuable fruit crops, is broadly planted in the world. As reported by FAO, the annual world production of grape in 2013 exceeded 77 million tones, China, however, ranks the first place with the production over 10 million tones.To date, one of the real challenges for grape industry under the background of continuing increasement of planting area worldwide is, how to reduce the risk, as well as how to develop the best management of pathogen infection. Grape is under the threat of many pathogens from bacteria, fungi and virus. The powdery mildew(PM) is one of the most prevalent fungal diseases in grape, which causes great losses in grape producing areas. Unfortunately, the European grapevine(Vitis vinifera), which is a common grape species for either wine making or fresh consuming, is susceptible to PM. Therefore, development of PM(and other diseases)resistant plant is the key task for grape breeding field. In China, great progress has been made on evaluating the PM resistance in diverse sources of wild grapes. These pioneering work unraveled many valuable characteristics of Chinese wild grape, however, the underlying mechanisms are largely unknown. The principle goal for this study is to explore the molecular mechanism for PM resistance in Chinese wild grape, to achieve this, we collected three wild grapes with distinguished PM resistance at several time points during infection. The transcriptomes were profiled and assembled de novo. We compared our de novo assemblies with respect to the reference genome of PN40024, and revealed a bunch of structure variations. Meanwhile, the differentially expessed genes(DEGs) were identified from two grapes(one resistant and one susceptible accessions) at different infection stages. Furthermore,we conducted experiments to verify the biological functions of two resistance related proteins,BAK1 and LecRK1, which were initially identified in our previous experiments. To summarize, the overall results and significant conclusions are as follows:1. Three Chinese wild grapes, two PM resistant and one PM susceptible, were inoculatedwith PM, and collected at different time points during infection. We assembled de novo the transcriptomes for all accessions, and compared with respect to the reference genome PN40024. We reported that Chinese wild grapes possess abundant distinct genes and genetic variations, such as SNPs and small indels. For each accession, we identified more than 1,000 distinct genes and 600~700 non-coding transcripts, ca. 30% distinct genes were functionally connected with plant defense response. Specifically, biological processes, for example, the plant defense responses against pathogen infection and coumarin related phytoalexin biosynthesis, were mostly enriched in distinct genes. 6~10% of distinct genes were predicted as receptor-like kinases(RLK), and 8% were NBS-LRR resistant genes. RLK and NBS-LRR are two groups of genes that functionalize in recognizing pathogens and signaling at different stages. The enrichment of these genes, which are directly or indirectly interacted with pathogens, may implicate the selection during pathogen-host coevolution. Moreover, we also identified 87,000-110,000 homozygous SNPs and ~2,000 small indels between wild grapes and the reference. We evaluated the potential impact of these variations on the consequence of protein encoding. Finally, by the aid of strand-specific information, we found 100-200 cisNAT pairs, these transcripts were overrepresented in the secondary metabolism and abiotic stresses, which implies a potential role for cis-NAT regulation.2. Apart from structure variations, we want to know what genes might be differently regulated at transcription level across accessions and infection stages. To this end, we collected the tissues in different infection stages from one PM resistant and one PM susceptible wild grapes. Among all combinations of comparison, we identified totally 6,953 differentially expressed genes. Gene clustering analysis revealed that a considerable part of gene clusters showed consistent expression pattern between two grapes, suggesting the regulatory conservation for these genes. However, we did observed a class of important resistance-related genes, which showed distinct expression patterns. R genes containing NBS domain, were repressed overtime in PM susceptible accession, while others containing receptor domains were significantly induced in PM resistant accession. GO enrichment analysis revealed many disease resistance related processes were significantly suppressed in PM susceptible accession during early infection. Moreover, common disease related metabolisms were enriched in upregulated genes in both accessions, while some rare reported primary or secondary metabolism pathways showed inconsistent enrichment in two accessions.3. Rather than systematic view of genetic variations, we also conducted experimental verification of some important proteins involved in PM resistance in grape. For example,from previous mRNA differential display PCR experiments, we obtained an EST sequencethat associated with PM resistance in grape. This EST was induced in both PM resistant and susceptible accessions, but the expression patterns were not consistent. Cloning and sequencing the full length ORF of this EST enables us to identify the domains encoded, which are, the G-type lectin domain, transmembrane domain, and ser/thr protein kinase domain.Therefore, we classified this gene as lectin receptor-like kinase and named VqLecRK1. The subcellular localization of VqLecRK1 was further confirmed by GFP co-localization in Arabidopsis. Functional analysis of VqLecRK1 by heterogeneous expression of this gene in Arabidopsis showed increased resistance to both PM and Pst DC3000. The defence responses for biotrophic pathogens, such as callose deposition, ROS burst and cell death, were obvious in the VqLecRK1 overexpression Arabidopsis. In addition, inoculation with Pst DC3000 and treated with PAMPs in VqLecRK1 overexpression lines showed increased PTI responses, but the magnitude of callose deposition and stomatal changes were variable. These data suggested that VqLecRK1 involves in defence response.4. One of the side discoveries in the aforementioned transcriptomes is the induced expression for SERK family upon PM infection. However, different members in SERK family showed variable expression patterns, of these, VqBAK1/VqSERK3 showed the hightest expression value and prominent expression increase. We thus cloned this gene, and confirmed its subcellular localization using GFP co-localization in Arabidopsis. Heterogeneous expression of VqBAK1 in Arabidopsis bak1-4 mutant recovered the control of aberrant cell death due to the lack of At BAK1, proved that VqBAK1 also control cell death in grape.Furthermore, we found that overexpression of VqBAK1 in Arabidopsis increased the resistance to Pst DC3000, as well as enhanced PTI upon treatment with PAMPs. Collectively,we demonstrated that VqBAK1 is functional conserved in controlling cell death and enhancing PTI signal across species. |
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