Plant pathogens, such as bacteria, fungi, oomycetes and nematodes, rely on wide range of virulent effectors delivered into host cells to suppress plant immunity. Although phytobacterial effectors have been intensively investigated, little is known about the function of effectors of plant-parasitic nematodes, such as Globodera pallida , a cyst nematode responsible for vast losses in the potato and tomato industries. Here, we demonstrate using in vivo and in vitro ubiquitination assays the potato cyst nematode ( Globodera pallida ) effector RHA1B is an E3 ubiquitin ligase that employs multiple host plant E2 ubiquitin conjugation enzymes to catalyze ubiquitination. RHA1B was able to suppress effector-triggered immunity (ETI), as manifested by suppression of hypersensitive response (HR) mediated by a broad range of nucleotide-binding leucine-rich repeat (NB-LRR) immune receptors, presumably via E3-dependent degradation of the NB-LRR receptors. RHA1B also blocked the flg22-triggered expression of Acre31 and WRKY22 , marker genes of pathogen‐associated molecular pattern (PAMP)‐triggered immunity (PTI), but this did not require the E3 activity of RHA1B. Moreover, transgenic potato overexpressing the RHA1B transgene exhibited enhanced susceptibility to G . pallida . Thus, our data suggest RHA1B facilitates nematode parasitism not only by triggering degradation of NB-LRR immune receptors to block ETI signaling but also by suppressing PTI signaling via an as yet unknown E3-independent mechanism.

The root-knot nematode (RKN) Meloidogyne hispanica has been found in all continents associated with a wide host range, including economically, important plants and can be considered a species of emerging importance. Considerable progress has been made to identify nematode effector genes as they are important targets for the development of novel control strategies. The effector genes, venom allergen-like protein (vap-1) and fatty acid and retinol binding protein (far-1), were identified, isolated and sequenced in M. hispanica (Mhi-vap-1 and Mhi-far-1) using the genome information available for the RKNs M. incognita and M. hapla. These genes are differentially expressed during M. hispanica development and their amplification products were observed from cDNA of the eggs, second-stage juveniles (J2) and adult females.However, Mhi-vap-1 showed the highest level of expression in J2. In situ hybridization analysis revealed that the Mhi-vap-1 and Mhi-far-1 transcripts are accumulated within the J2 subventral oesophageal glands. The specific expression in the subventral oesophagel glands and presence of the secretion signal peptide for both genes suggests that these proteins are secreted by the J2 and may play a role in the early parasitic stage of the infection process. These genes were also isolated and sequenced in M. arenaria, M. incognita and M. javanica; and phylogenetic analysis revealed that the predicted protein sequences belonging to M. hispanica and several other species of plant-parasitic nematodes have a high degree of conservation.

Meloidogyne hispanica (Mhi) is a difficult-to-control polyphagous root-knot nematode (RKN) species of emerging importance for economically valuable crops. Nematode secretions are likely to be the first signals perceived by the plant and are thought to be involved in various aspects of the plant-nematode interaction. The aims of this work were to identify and characterize M. hispanica parasitism genes: cathepsin L cysteine protease (cpl-1), calreticulin (crt-1), β-1,4-endoglucanase-1 (eng-1) and manganese superoxide dismutase (mnsod). As there are no genomic data available for M. hispanica, primers were designed from the conserved regions of the putative parasitism genes in M. incognita and M. hapla and used to amplify the genes in M. hispanica, which led to the successful amplification of these genes in M. hispanica. Partial gene sequences were also obtained for M. arenaria, M. hapla, M. hispanica, M. incognita and M. javanica cpl-1, crt-1, eng-1 and mnsod genes, and their phylogenetic relationship analysed. In order to determine whether these genes are differentially expressed during M. hispanica development, cDNA was amplified from mRNA isolated from eggs, second-stage juveniles (J2) and females. Amplification products were observed from cDNA of all developmental stages for the Mhi-cpl-1 and Mhi-crt-1 genes. However, the gene Mhi-crt-1 exhibited intense amplification bands in females, while the Mhi-eng-1 gene was equally amplified in eggs and J2 and the Mhi-mnsod gene was only expressed in eggs. In comparison to the other RKN species, the genes Mhi-eng-1 and Mhi-mnsod showed transcription in different nematode developmental stages.

Gene silencing using RNAi is a powerful tool for functional analysis of nematode genes and can provide a new strategy for the management of root‐knot nematodes. The transcript of the venom allergen‐like protein gene (Mhi‐vap‐1) of Meloidogyne hispanica is localized in the subventral oesophageal gland cells of second‐stage juveniles (J2) and the gene is highly transcribed in this developmental nematode stage. The purpose of this study was to assess whether the silencing of the Mhi‐vap‐1 gene could affect nematode attraction to roots, penetration, development and reproduction in tomato plants. The optimum soaking time to generate silencing of this gene was determined by quantitative RT‐PCR analysis of the relative expression of the Mhi‐vap‐1 gene in the J2 incubated with the soaking solution for 24, 32 and 48 h. At 48 h, the relative expression of Mhi‐vap‐1 decreased, which indicates that this period of time is optimum to silence this nematode gene using the RNAi method. Silencing of the Mhi‐vap‐1 gene interfered with the completion of the nematode life cycle and caused a reduction in nematode attraction to roots, penetration and infection of plants. A small difference in the number of females and galls formed was also observed, which caused a small decrease in the nematode reproduction factor. The use of RNAi silencing of the Meloidogyne effector gene Mhi‐vap‐1 showed that this gene is important for the plant–nematode interaction during the early events of infection and could be a target gene for anti‐nematode strategies.