| Triticum aestivum L.is one of the most important crop species and accounts for 21% of the food calories to more than 4.5 billion people throughout the world.The most globally recognized and economically important wheat nematodes are the cereal cyst nematodes(CCNs;Heterodera avenae,H.filipjevi,and H.latipons),H.avenae and H.filipjevi are the two main CCN species occurring,and have been found to infect cereals in 16 provinces in China.CCN,a major limitation of wheat production,is widespread in most wheat-growing regions in China.In China,more than 4 million hectares in major wheat-producing regions are infected by CCN,with losses estimated at 1.9 billion RMB per year.Plant parasitic nematodes(PPNs)can soften and/or degrade the host cell walls,suppress or prevent host defense responses and manipulate host signaling pathways to generate complex feeding structures.Although putative novel effectors have been identified by genomic,transcriptomic and proteomic methods,little is known about the molecular details of CCNs parasitism.Here,we performed a high-throughput sequencing analysis of the H.avenae and H.filipjevi transcriptome at five and six developmental stages.For H.avenae and H.filipjevi,a total of 82,549/106,755 unigenes were ultimately obtained,and 747/885 transcripts had best hits to genes putatively encoding carbohydrate-active enzymes in plant parasitic nematodes that play an important role in the invasion process.A total of 1,634/1,706 transcripts showing high homology to known phytonematode effectors and 63/87 putative novel effectors were identified in the H.avenae transcriptomes.Furthermore,115 and 107 transcripts were found to have putative lethal RNA interference(RNAi)phenotypes in the H.avenae and H.filipjevi transcriptome.Twenty-three and twenty-four unigenes were analyzed by qRT-PCR and confirmed to be highly expressed during at least one developmental stage in H.avenae and H.filipjevi.Twenty-one of the 24 tested putative novel effectors were specifically expressed and located in the cells of the subventral glands or dorsal glands.Three target genes with lethal RNAi phenotypes and two of the four tested putative effectors were associated with a decrease in the number of cysts using in vitro RNAi technology.In this study,we screened 51 wheat lines(CIMMYT)carrying different Cre genes(Cre1,Cre2,Cre3,Cre5,Cre7,Cre8,CreR and Pt)from 2012-2014,and identified one and five cultivars that were immune(M)and resistant(R),respectively,to H.avenae pathotype Ha91.The Cre3 gene conferred the most effective resistance against H.avenae pathotype Ha91 in both field and greenhouse assays.Conversely,the Cre1,Cre2,Cre7,Cre8 and CreR genes conferred the lowest effective resistance.Using Pluronic F-127 gel and a staining assay,juvenile nematodes invading wheat roots were observed,and nematode development was analyzed.Compared to R and M roots,those of the susceptible(S)wheat cultivar were more attractive to H.avenae second-stage juveniles(J2s).Similarly,roots of both S and R cultivars are easily invaded by H.avenae J2 s.We observed the retardation of nematode development in R cultivars and tiny white female cysts protruding from the R cultivar.Nematodes in M roots either disintegrated or remained J2 s or third-stage juveniles(J3s)and failed to complete their life cycle.Molting was also suppressed or delayed in R and M genotypes.Nonetheless,there are large differences in syncytial composition between R and susceptible(S)CCN cultivars.For both S and R cultivars,syncytia were characterized by cell wall perforations and dense cytoplasm in hypertrophied syncytium component cells.Syncytial size increased gradually with nematode development in S cultivars.Moreover,an incompatibility reaction occurred in M wheat roots: the syncytium was disorganized,exhibiting disintegration and condensed nuclei. |
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