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.

The plant-parasitic nematode Meloidogyne incognita secretes MIF-like proteins into plant tissues, and MiMIF-2 interacts with two plant annexins to suppress plant immune responses and promote parasitism.

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Root-knot nematodes, Meloidogyne spp., are obligate endoparasites that maintain a biotrophic relationship with their hosts. They infect roots as microscopic vermiform second-stage juveniles, and establish specialized feeding structures called 'giant-cells', from which they withdraw water and nutrients. The nematode effector proteins secreted in planta are key elements in the molecular dialogue of parasitism. Here, we compared Illumina RNA-seq transcriptomes for M. incognita obtained at various points in the lifecycle, and identified 31 genes more strongly expressed in parasitic stages than in preparasitic juveniles. We then selected candidate effectors for functional characterization. Quantitative real-time PCR and in situ hybridizations showed that the validated differentially expressed genes are predominantly specifically expressed in oesophageal glands of the nematode. We also soaked the nematodes in siRNA to silence these genes and to determine their role in pathogenicity. The silencing of the dorsal gland specific-Minc18876 and its paralogues resulted in a significant, reproducible decrease in the number of mature females with egg masses, demonstrating a potentially important role for the small glycine- and cysteine-rich effector MiSGCR1 in early stages of plant-nematode interaction. Finally, we report that MiSGCR1 suppresses plant cell death induced by bacterial or oomycete triggers of plant defense.

Large amounts of effectors are secreted by the oesophageal glands of plant-parasitic nematodes, but their molecular mode of action remains largely unknown. We characterized a Meloidogyne incognita protein disulphide isomerase (PDI)-like effector protein (MiPDI1) that facilitates nematode parasitism. In situ hybridization showed that MiPDI1 was expressed specifically in the subventral glands of M. incognita. It was significantly upregulated during parasitic stages. Immunolocalization demonstrated MiPDI1 secretion in planta during nematode migration and within the feeding cells. Host-induced silencing of the MiPDI1 gene affected the ability of the nematode to infect the host, whereas MiPDI1 expression in Arabidopsis increased susceptibility to M. incognita, providing evidence for a key role of MiPDI1 in M. incognita parasitism. Yeast two-hybrid, bimolecular fluorescence complementation and coimmunoprecipitation assays showed that MiPDI1 interacted with a tomato stress-associated protein (SlSAP12) orthologous to the redox-regulated AtSAP12, which plays an important role in plant responses to abiotic and biotic stresses. SAP12 silencing or knocking out in Nicotiana benthamiana and Arabidopsis increased susceptibility to M. incognita. Our results suggest that MiPDI1 acts as a pathogenicity factor promoting disease by finetuning SAP-mediated responses at the interface of redox signalling, defence and stress acclimation in Solanaceae and Arabidopsis.

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Bacillus cereus strain Bc-cm103 shows nematicidal activity and therefore has been used as a biological control agent to control the root-knot nematode Meloidogyne incognita. However, it remains unknown whether volatile organic compounds (VOCs) produced by B. cereus strain Bc-cm103 are effective in biocontrol against M. incognita. Therefore, in this study, we investigated the activity of Bc-cm103 VOCs against M. incognita. The B. cereus strain Bc-cm103 significantly repelled the second-stage juveniles (J2s) of M. incognita. In vitro evaluation of VOCs produced by the fermentation of Bc-cm103 in a three-compartment Petri dish revealed the mortality rates of M. incognita J2s as 90.8% at 24 h and 97.2% at 48 h. Additionally, evaluation of the ability of Bc-cm103 VOCs to suppress M. incognita infection in a double-layered pot test showed that root galls on cucumber roots decreased by 46.1%. Furthermore, 21 VOCs were identified from strain Bc-cm103 by solid-phase micro-extraction gas chromatography-mass spectrometry (SPME-GC-MS), including alkanes, alkenes, esters and sulfides. Among them, dimethyl disulfide (30.63%) and S-methyl ester butanethioic acid (30.29%) were reported to have strong nematicidal activity. Together, these results suggest that B. cereus strain Bc-cm103 exhibits fumigation activity against M. incognita.

Meloidogyne incognita is obligate parasitic nematode with a wide variety of hosts that causes huge economic losses every year. In an effort to identify novel bacterial biocontrols against M. incognita, the nematicidal activity of Bacillus velezensis strain Bv-25 obtained from cucumber rhizosphere soil was measured. Strain Bv-25 could inhibit the egg hatching of M. incognita and had strong nematicidal activity, with the mortality rate of second-stage M. incognita juveniles (J2s) at 100% within 12 h of exposure to Bv-25 fermentation broth. The M. incognita genes ord-1, mpk-1, and flp-18 were suppressed by Bv-25 fumigation treatment after 48 h. Strain Bv-25 could colonize cucumber roots, with 5.94 × 107 colony-forming units/g attached within 24 h, effectively reducing the infection rate with J2s by 98.6%. The bacteria up-regulated the expression levels of cucumber defense response genes pr1, pr3, and lox1 and induced resistance to M. incognita in split-root trials. Potted trials showed that Bv-25 reduced cucumber root knots by 73.8%. The field experiment demonstrated that disease index was reduced by 61.6%, cucumber height increased by 14.4%, and yield increased by 36.5% in Bv-25–treated plants compared with control. To summarize, B. velezensis strain Bv-25 strain has good potential to control root-knot nematodes both when colonizing the plant roots and through its volatile compounds.