Heng Jian

Msp40 effector of root-knot nematode manipulates plant immunity to facilitate parasitism.

Root-knot nematodes (RKNs) are obligate biotrophic parasites that invade plant roots and engage in prolonged and intimate relationships with their hosts. Nematode secretions, some of which have immunosuppressing activity, play essential roles in successful parasitism; however, their mechanisms of action remain largely unknown. Here, we show that the RKN-specific gene MiMsp40, cloned from Meloidogyne incognita, is expressed exclusively in subventral oesophageal gland cells and is strongly upregulated during early parasitic stages. Arabidopsis plants overexpressing MiMsp40 were more susceptible to nematode infection than were wild type plants. Conversely, the host-derived MiMsp40 RNAi suppressed nematode parasitism and/or reproduction. Moreover, overexpression of MiMsp40 in plants suppressed the deposition of callose and the expression of marker genes for bacterial elicitor elf18-triggered immunity. Transient expression of MiMsp40 prevented Bax-triggered defence-related programmed cell death. Co-agroinfiltration assays indicated that MiMsp40 also suppressed macroscopic cell death triggered by MAPK cascades or by the ETI cognate elicitors R3a/Avr3a. Together, these results demonstrate that MiMsp40 is a novel Meloidogyne-specific effector that is injected into plant cells by early parasitic stages of the nematode and that plays a role in suppressing PTI and/or ETI signals to facilitate RKN parasitism.

RNAi silencing of the Meloidogyne incognita Rpn7 gene reduces nematode parasitic success

RNA interference (RNAi) techniques provide a major breakthrough in functional analysis for plant parasitic nematodes (PPNs). It offers the possibility of identifying new essential targets and consequently developing new resistance transgenes. To validate the potential of Mi-Rpn7 as a target for controlling root knot nematode Meloidogyne incognita and to evaluate the feasibility of our modified platform for assessing silencing phenotypes, we knocked down the Rpn7 gene of M. incognita using RNAi in vitro and in vivo. After soaking with 408-bp Rpn7 dsRNA, pre-parasitic second-stage juvenile (J2) nematodes showed specific transcript knockdown, resulting in an interrupted locomotion in an attraction assay with Pluronic gel medium, and consequently in a reduction of nematode infection ranging from 55.2% to 66.5%. With in vivo expression of Rpn7 dsRNA in transformed composite plants, the amount of egg mass per gram root tissue was reduced by 34% (P < 0.05) and the number of eggs per gram root tissue was reduced by 50.8% (P < 0.05). Our results demonstrated that the silencing of the Rpn7 gene in M. incognita J2s significantly reduced motility and infectivity. Although it does not confer complete resistance, Mi-Rpn7 RNAi in hairy roots produced significant negative impacts on reproduction and motility of M. incognita. In addition, the presented modified procedure provides technique reference for PPN genes functional analysis or target screening.

An ANNEXIN-like protein from the cereal cyst nematode Heterodera avenae suppresses plant defense.

Parasitism genes encoding secreted effector proteins of plant-parasitic nematodes play important roles in facilitating parasitism. An annexin-like gene was isolated from the cereal cyst nematode Heterodera avenae (termed Ha-annexin) and had high similarity to annexin 2, which encodes a secreted protein of Globodera pallida. Ha-annexin encodes a predicted 326 amino acid protein containing four conserved annexin domains. Southern blotting revealed that there are at least two homologies in the H. avenae genome. Ha-annexin transcripts were expressed within the subventral gland cells of the pre-parasitic second-stage juveniles by in situ hybridization. Additionally, expression of these transcripts were relatively higher in the parasitic second-stage juveniles by quantitative real-time RT-PCR analysis, coinciding with the time when feeding cell formation is initiated. Knockdown of Ha-annexin by method of barley stripe mosaic virus-based host-induced gene silencing (BSMV-HIGS) caused impaired nematode infections at 7 dpi and reduced females at 40 dpi, indicating important roles of the gene in parasitism at least in early stage in vivo. Transiently expression of Ha-ANNEXIN in onion epidermal cells and Nicotiana benthamiana leaf cells showed the whole cell-localization. Using transient expression assays in N. benthamiana, we found that Ha-ANNEXIN could suppress programmed cell death triggered by the pro-apoptotic mouse protein BAX and the induction of marker genes of PAMP-triggered immunity (PTI) in N. benthamiana. In addition, Ha-ANNEXIN targeted a point in the mitogen-activated protein kinase (MAPK) signaling pathway downstream of two kinases MKK1 and NPK1 in N. benthamiana.

Cloning and characterization of a venom allergen-like protein gene cluster from the pinewood nematode Bursaphelenchus xylophilus

Pinewood nematode (PWN) is the causal agent of the pine wilt disease. Previous studies have suggested that secretions from the esophageal glands of PWN play an important role in pathogenicity. A cluster of three venom allergen-like protein genes and one pseudogene, Bx-vap-1, Bx-vap-2, Bx-vap-3 and Bx-vap-P, were identified within a 3.7-kb region. Additionally, three putative modification, transport and regulatory protein genes were also detected in the same flanking region of the Bx-vap gene cluster. Genes vap-1, -2 and -3 are functional and encode three major allelic variants of PWN venom allergen-like proteins. But Bx-vap-P is an untranscribed pseudogene. Genes vap-1, -2 and -3 produce predicted products of 204, 206 and 203 amino acid residues, respectively, including the putative signal peptide sequence at the amino termini. In situ mRNA hybridization analysis showed that the transcripts of genes vap-1, -2 and -3 accumulated exclusively within the esophageal gland cells of Bursaphelenchus xylophilus.

De Novo Analysis of the Transcriptome of Meloidogyne enterolobii to Uncover Potential Target Genes for Biological Control

Meloidogyne enterolobii is one of the obligate biotrophic root-knot nematodes that has the ability to reproduce on many economically-important crops. We carried out de novo sequencing of the transcriptome of M. enterolobii using Roche GS FLX and obtained 408,663 good quality reads that were assembled into 8193 contigs and 31,860 singletons. We compared the transcripts in different nematodes that were potential targets for biological control. These included the transcripts that putatively coded for CAZymes, kinases, neuropeptide genes and secretory proteins and those that were involved in the RNAi pathway and immune signaling. Typically, 75 non-membrane secretory proteins with signal peptides secreted from esophageal gland cells were identified as putative effectors, three of which were preliminarily examined using a PVX (pGR107)-based high-throughput transient plant expression system in Nicotiana benthamiana (N. benthamiana). Results showed that these candidate proteins suppressed the programmed cell death (PCD) triggered by the pro-apoptosis protein BAX, and one protein also caused necrosis, suggesting that they might suppress plant immune responses to promote pathogenicity. In conclusion, the current study provides comprehensive insight into the transcriptome of M. enterolobii for the first time and lays a foundation for further investigation and biological control strategies.

Large-Scale Identification and Characterization of Heterodera avenae Putative Effectors Suppressing or Inducing Cell Death in Nicotiana benthamiana

Heterodera avenae is one of the most important plant pathogens and causes vast losses in cereal crops. As a sedentary endoparasitic nematode, H. avenae secretes effectors that modify plant defenses and promote its biotrophic infection of its hosts. However, the number of effectors involved in the interaction between H. avenae and host defenses remains unclear. Here, we report the identification of putative effectors in H. avenae that regulate plant defenses on a large scale. Our results showed that 78 of the 95 putative effectors suppressed programmed cell death (PCD) triggered by BAX and that 7 of the putative effectors themselves caused cell death in Nicotiana benthamiana. Among the cell-death-inducing effectors, three were found to be dependent on their specific domains to trigger cell death and to be expressed in esophageal gland cells by in situ hybridization. Ten candidate effectors that suppressed BAX-triggered PCD also suppressed PCD triggered by the elicitor PsojNIP and at least one R-protein/cognate effector pair, suggesting that they are active in suppressing both pattern-triggered immunity (PTI) and effector-triggered immunity (ETI). Notably, with the exception of isotig16060, these putative effectors could also suppress PCD triggered by cell-death-inducing effectors from H. avenae, indicating that those effectors may cooperate to promote nematode parasitism. Collectively, our results indicate that the majority of the tested effectors of H. avenae may play important roles in suppressing cell death induced by different elicitors in N. benthamiana.

Field evaluation of Streptomyces rubrogriseus HDZ-9-47 for biocontrol of Meloidogyne incognita on tomato

Abstract Streptomyces rubrogriseus HDZ-9-47, isolated from eggs of Meloidogyne spp., was evaluated as a potential biocontrol agent of Meloidogyne incognita under in vitro and protective field. Microscopic observations showed that HDZ-9-47 parasitized eggs of M. incognita within 7 days. In vitro, the culture filtrate of HDZ-9-47 caused 97.0% mortality of second-stage juveniles (J2s) of M. incognita and inhibited more than 50% egg hatching. In the field, compared with the control, the root-knot index and J2s density in the treatment of drench the broth contained 1012 HDZ-9-47 spores were respectively reduced by 51.1 and 80.7% at 90 days post transplantation, which were better than that in other application doses and methods. In addition, reduction rates of root-knot index and J2s density of the treatment of combined application of HDZ-9-47 with biofumigation was 87.1 and 91.0%, respectively, better than either of HDZ-9-47 or biofumigation used alone or fosthiazate treatment. And tomato yield also increased by 16.1%. Together, our results suggest that HDZ-9-47 could be an effective biocontrol agent of M. incognita, and that application of HDZ-9-47 combined with cabbage residue biofumigation was a promising and sustainable option for M. incognita control.