Characterization of Putative Effectors from the Cereal Cyst Nematode Heterodera avenae

Abstract

Few molecular details of effectors of Heterodera avenae parasitism are known. We performed a high-throughput sequencing analysis of the H. avenae transcriptome at five developmental stages. A total of 82,549 unigenes were ultimately obtained, and 747 transcripts showed 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 1,480 unigenes were homologous to known phytonematode effectors, and 63 putative novel effectors were identified in the H. avenae transcriptomes. Twenty-three unigenes were analyzed by qRT-PCR and confirmed to be highly expressed during at least one developmental stage. For in situ hybridization, 17 of the 22 tested putative effectors were specifically expressed and located in the subventral gland cells, and five putative novel effectors were specifically expressed in the dorsal gland. Furthermore, 115 transcripts were found to have putative lethal RNA interference (RNAi) phenotypes. 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 through in vitro RNAi technology. These transcriptomic data lay a foundation for further studies of interactions of H. avenae with cereal and H. avenae parasitic control.

RNAi-Based Biocontrol of Wheat Nematodes Using Natural Poly-Component Biostimulants

“…Now that it has been established that biostimulants do have an anti-nematode effect, we look into whether this can be attributed to wheat plants producing additional si/miRNA complementary to mRNA of infested plants that is associated with plant genes, whose expression is upregulated during the entry of nematodes into plant cells, thus facilitating infestation of plants with nematodes, or to nematode mRNA associated with nematode genes that control nematode life cycle, nematode housekeeping genes, nematode parasitism or effector genes that are expressed during nematode entry into plant cells (Tsygankova et al, 2012a, 2014b; Li et al, 2014; Chen et al, 2015, 2017; Xu et al, 2016; Ali et al, 2017b; Yang et al, 2017; Cui et al, 2018).…”

“…Interestingly, for Avercom and Avercom nova-2, the level of enhancement of synthesis of si/miRNA complementary with nematode mRNA is higher than analogous levels for si/miRNA homologous with plant mRNA, while for Phytovit the level of synthesis of si/miRNA complementary with nematode mRNA is smaller than the respective level for plant mRNA. Comparison with Figure 2 indicates that while the synthesis of complementary si/miRNA can be enhanced very significantly, the difference between levels of reduction in nematode infestation is much smaller, suggesting that out of the total si/miRNA produced in the plant, only a part of it contained in the feeding cells formed on plant roots can enter nematode through ingestion using a stylet (Maule et al, 2011; Tsygankova et al, 2012a; Cui et al, 2018), possibly resulting in the subsequent nematode death.…”

“…We studied the impact of biostimulants on production within plant cells of si/miRNA that target both plant mRNA associated with plant host genes, whose expression is upregulated during nematode infestation, thus promoting penetration of nematodes into plant roots (Safari et al, 2005; Tsygankova et al, 2012a, 2014a; Kong et al, 2015; Ali et al, 2017a; Chen et al, 2017), and these can be used as target genes for silencing (Koch and Kogel, 2014), and nematode mRNA associated with genes that control nematode life cycle, nematode housekeeping genes, nematode parasitism, or effector genes that are expressed during nematode entrance into plant cells (Tsygankova et al, 2012a, 2014a; Li et al, 2014; Chen et al, 2015, 2017; Xu et al, 2016; Ali et al, 2017b; Yang et al, 2017; Cui et al, 2018). For this purpose we perform dot blot hybridization of [ 33 P]-labeled si/miRNA isolated from experimental plants with mRNA isolated from control plants, i.e., plants uninfested by nematodes and not treated by biostimulants, as well as with mRNA molecules isolated from nematode larvae.…”

“…Yang et al (2017) have sequenced mRNA from both pre- and post-parasitic stages of H. avenae , providing an extensive characterization of development, metabolism, and parasitism genes. A recent work by Cui et al (2018) has identified three specific H. avenae genes c 59821_ g 1, c 69968_ g 1, and c 45915_ g 1 associated with lethal phenotypes, which suggests that they can be used as effective genetic targets for plant-induced RNAi. Similarly, Zheng et al (2015) have studied transcriptome of second-stage juveniles of H. avenae and shown that silencing unigene38116, unigene102492, and unigene38007 genes, which are homologs of C. elegans genes C 18 D 1.3, F 38 E 11.7, and C 06 G 4.2, respectively, by soaking nematode larvae in the gene-specific siRNA has lethal effect on the nematode.…”

Two venom allergen‐like proteins, HaVAP1 and HaVAP2, are involved in the parasitism of Heterodera avenae

“…In this study, we isolated and cloned the full‐length cDNA and gDNA of two VAP genes, HaVAP1 and HaVAP2 , from H. avenae based on an EST library (Cui et al. , ). To explore the functions of these two genes during H. avenae parasitism, experiments were carried out and the results are discussed below.…”