| Cereal cyst nematode(CCN) is one of the most important plant pathogens of wheat worldwide, posing a high threat on wheat production. Plant nematodes secret effector proteins towards host plant cells by their stylet, resulting in successful nematode parasitism and plant disease. But the researches of molecular interaction between CCN and its host seem to be very lacking and backward so far mainly because of a lack of an effective and applicable pathosystem.In this study, we cloned a putative CCN effector gene by RACE according to transcriptional data. The full length for this gene c DNA is 837 bp, almong which owns a 585 bp ORF, encoding one 194-amino-acids peptide. In N-end of this gene, there is a 21-amino-acids signal peptide. In addition, we found no transmembrane domain within this gene. By BLAST, we found this gene had a high similarity with G20E03 gene in Heterodera glycines. Therefore we named this Ha-G20E03. By in situ hybirdation, we found the transcripts of Ha-G20E03 accumulated specifically in the gland cells of H. avenae, indicating that Ha-G20E03 may play a vital role in H. avenae parasitism.In order to better understand the function of this gene, cereal model plant Brachypodium distachyon, diploid wheat 2A and 2D were considered as the candidate cereal model plant for CCN to replace wheat to investigate the CCN-host interactions. In this study, by CCN inoculation assay, ROS content measurement, q RT-PCR, histological analysis of the syncytia and barley stripe mosaic virus-induced gene silencing(BSMV-VIGS), we assayed the compatibility between CCN and these three cereal candidate plants, and assessed possible reason for this incompatibility and did silencing study in the compatible plant. This study indicated that CCN could infest into the roots of Bd21-3 but fail to develop into the following stages and complete the life cycle, and this incompatibility was not dependent on the CCN population difference. In addition, we found there was a strong ROS burst at 3 days post inoculation, and at this time point, seven Class III peroxidase genes and two NADPH Oxidase genes were significantly up-regulated after CCN inoculation. We then found CCN could both infest into the roots of G1812 and AL8/78 and develop into J3, J4 and adult female.Moreover, the developmental processes and percentage of each CCN stage in test plants were quite similar with those in susceptible wheat. Histological analysis of the syncytia in diploid wheat induced by CCN showed the syncytia in 2D showed a little smaller. The number of cyst formed in 2A is fairly similar with that formed in control. By contrast, 2D showed a somewhat resistance to CCN. We also established a system of BSMV-VIGS in our lab. We successfully silenced the target gene PDS in wheat and the test wheat showed photobleaching effects. In addition, we selected Jimai22 that has the best silencing effect as host wheat for VIGS assays in the future. Furthermore, the study of a system of BSMV-VIGS in diploid wheat 2A is still ongoing.In conclusion, in this study, we cloned Ha-G20E03 from H. avenae, and revealed that the transcripts of Ha-G20E03 accumulated specifically in the gland cells of H. avenae. All of these could contribute to a better understanding of CCN-host interactions. We also confirmed B. distachyon is a non-host for CCN and failed to be severed as a model plant for CCN-host interactions. Moreover, we found ROS burst may be an essential reason for this incompatibility and POX genes may play a key role for it. By contrast, both diploid wheat 2A and 2D was compatible with CCN. 2A showed more susceptible to CCN, indicating that 2A have a strong potential to be a cereal model plant in research of CCN-host interactions. |
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