Resistance-breaking population of Meloidogyne incognita utilizes plant peroxidase to scavenge reactive oxygen species, thereby promoting parasitism on tomato carrying Mi-1 gene.

Resistance conferred by the Mi-1 gene from Solanum peruvianum is effective and widely used for controlling root-knot nematodes (RKNs, Meloidogyne spp.). However, breakdown of resistance by RKNs seriously threatens the durable application of the resistance resource. Here, a resistance-breaking population of M. incognita was selected from an avirulent population by continuously inoculating on Mi-1-carrying tomato. Histological observations showed the resistance-breaking population would not induce hypersensitive response (HR) when infecting Mi-1-carrying tomato, while avirulent population did. A total of 308 differentially expressed genes (DEGs) were identified from Mi-1-carrying tomato upon infection with resistance-breaking versus avirulent populations by RNA-seq. The expression patterns of 23 selected DEGs were validated by quantitative real-time PCR (qRT-PCR). Subsequently, seven out of nine highly up-regulated DEGs were successfully knocked down in Mi-1-carrying tomato by tobacco rattle virus (TRV) mediated RNAi. The TRV line targeting a peroxidase gene showed a much higher magnitude of reactive oxygen species (ROS) and distinct reduction of pathogenicity upon infection of the resistance-breaking population compared with that of TRV::gfp line. Our results suggested that plant peroxidase might be exploited by resistance-breaking population of M. incognita to scavenge ROS, so as to overcome Mi-1-mediated resistance.

Versatile glycoside hydrolase family 18 chitinases for fungi ingestion and reproduction in the pinewood nematode Bursaphelenchus xylophilus.

The glycoside hydrolase family 18 (GH18) of chitinases is a gene family widely expressed in archaes, prokaryotes and eukaryotes, and hydrolyzes the ß-1,4-linkages in chitin. The pinewood nematode Bursaphelenchus xylophilus is one of the organisms that produces GH18 chitinases. Notably, B. xylophilus has a higher number of GH18 chitinases compared with the obligate plant-parasitic nematodes Meloidogyne incognita and Meloidogyne hapla. In this study, seven GH18 chitinases were identified and cloned from B. xylophilus based on genomic analyses. The deduced amino acid sequences of all these genes contained an N-terminal signal peptide and a GH18 catalytic domain. Phylogenetic analysis showed that the origin of B. xylophilus GH18 chitinases was independent of those from fungi and bacteria. Real-time quantitative reverse transcription PCR analysis indicated that GH18 chitinase genes had discrete expression patterns, representing almost all the life stages of B. xylophilus. In situ hybridisation showed that the mRNA of GH18 chitinase genes of B. xylophilus were detected mainly in the spermatheca, esophageal gland cells, seminal vesicle and eggs. RNA interference (RNAi) results revealed different roles of GH18 chitinase genes in B. xylophilus. Bx-chi-1, Bx-chi-2 and Bx-chi-7 were associated with reproduction, fungal cell-wall degradation and egg hatching, respectively. Bx-chi-5 and Bx-chi-6 may be involved in sperm metabolism. In conclusion, this study demonstrates that GH18 chitinases have multiple functions in the life cycle of B. xylophilus.

Isolation and characterization of a fatty acid- and retinoid-binding protein from the cereal cyst nematode Heterodera avenae.

A gene encoding fatty acid- and retinoid-binding protein was isolated from the cereal cyst nematode Heterodera avenae and the biochemical function of the protein that it encodes was analysed. The full-length cDNA of the Ha-far-1 gene is 827 bp long and includes a 22- nucleotide trans-spliced leader sequence (SL1) at its 5-end. The genomic clone of Ha-far-1 consists of eight exons separated by seven introns, which range in size from 48 to 186 bp. The Ha-far-1 cDNA contains an open reading frame encoding a 191 amino acid protein, with a predicted secretory signal peptide. Sequence analysis showed that Ha-FAR-1 has highest similarity to the Gp-FAR-1 protein from the potato cyst nematode, Globodera pallida and that the protein was grouped with all homologues from other plant-parasitic nematodes in a phylogenetic analysis. Fluorescence-based ligand binding analysis confirmed that the recombinant Ha-FAR-1 protein was able to bind fatty acids and retinol. Spatial and temporal expression assays showed that the transcripts of Ha-far-1 accumulated mainly in the hypodermis and that the gene is most highly expressed in third-stage juveniles of H. avenae. Fluorescence immunolocalization showed that the Ha-FAR-1 protein was present on the surface of the infective second-stage juveniles of H. avenae. Nematodes treated with dsRNA corresponding to Ha-far-1 showed significantly reduced reproduction compared to nematodes exposed to dsRNA from a non-endogenous gene, suggesting that Ha-far-1 may be an effective target gene for control of H. avenae using an RNAi strategy.

Exposure to double-stranded RNA mediated by tobacco rattle virus leads to transcription up-regulation of effector gene Mi-vap-2 from Meloidogyne incognita and promotion of pathogenicity in progeny.

Meloidogyne spp. are economically important plant parasites and cause enormous damage to agriculture world-wide. These nematodes use secreted effectors which modify host cells, allowing them to obtain the nutrients required for growth and development. A better understanding of the roles of effectors in nematode parasitism is critical for understanding the mechanisms of nematode-host interactions. In this study, Mi-vap-2 of Meloidogyne incognita, a gene encoding a venom allergen-like protein, was targeted by RNA interference mediated by the tobacco rattle virus. Unexpectedly, compared with a wild type line, a substantial up-regulation of Mi-vap-2 transcript was observed in juveniles collected at 7 days p.i. from Nicotiana benthamiana agroinfiltrated with TRV::vap-2. This up-regulation of the targeted transcript did not impact development of females or the production of galls, nor the number of females on the TRV::vap-2 line. In a positive control line, the transcript of Mi16D10 was knocked down in juveniles from the TRV::16D10 line at 7 days p.i., resulting in a significant inhibition of nematode development. The up-regulation of Mi-vap-2 triggered by TRV-RNAi was inherited by the progeny of the nematodes exposed to double-stranded RNA. Meanwhile, a substantial increase in Mi-VAP-2 expression in those juvenile progeny was revealed by ELISA. This caused an increase in the number of galls (71.2%) and females (84.6%) produced on seedlings of N. benthamiana compared with the numbers produced by control nematodes. Up-regulation of Mi-vap-2 and its encoded protein therefore enhanced pathogenicity of the nematodes, suggesting that Mi-vap-2 may be required for successful parasitism during the early parasitic stage of M. incognita.