Characterization of Five Meloidogyne incognita Effectors Associated with PsoRPM3.

Root-knot nematodes (RKNs) are devastating parasites that invade thousands of plants. In this study, five RKN effectors, which might interact with Prunussogdiana resistance protein PsoRPM3, were screened and identified. In situ hybridisation results showed that MiCal, MiGST_N_4, MiEFh and MiACPS are expressed in the subventral oesophageal glands (SvG), and MiTSPc hybridization signals are found in the dorsal esophageal gland (DG) of Meloidogyne incognita in the pre-J2. RT-qPCR data indicated that the expression of MiCal, MiGST_N_4, MiEFh, and MiACPS genes are highly expressed in M. incognita of pra-J2 and J3/J4 stages. The expression of MiTSPc increased significantly in the female stage of M. incognita. Moreover, all effectors found in this study localize in the cytoplasm and nucleus when transiently expressed in plant cells. In addition, MiGST_N_4, MiEFh, MiACPS and MiTSPc can elicit the ROS burst and strong hypersensitive response (HR), as well as significant ion leakage. Our data suggest that MiGST_N_4, MiEFh, MiACPS and MiTSPc effectors may be involved in triggering the immune response of the host plant.

Overexpression of PsoRPM3, an NBS-LRR gene isolated from myrobalan plum, confers resistance to Meloidogyne incognita in tobacco.

KEY MESSAGES: We reported an NBS-LRR gene, PsoRPM3, is highly expressed following RKN infection, initiating an HR response that promotes plant resistance. Meloidogyne spp. are root-knot nematodes (RKNs) that cause substantial economic losses worldwide. Screening for resistant tree resources and identifying plant resistance genes is currently the most effective way to prevent RKN infestations. Here, we cloned a novel TIR-NB-LRR-type resistance gene, PsoRPM3, from Xinjiang wild myrobalan plum (Prunus sogdiana Vassilcz.) and demonstrated that its protein product localized to the nucleus. In response to Meloidogyne incognita infection, PsoRPM3 gene expression levels were significantly higher in resistant myrobalan plum plants compared to susceptible plants. We investigated this difference, discovering that the - 309 to - 19 bp region of the susceptible PsoRPM3 promoter was highly methylated. Indeed, heterologous expression of PsoRPM3 significantly enhanced the resistance of susceptible tobacco plants to M. incognita. Moreover, transient expression of PsoRPM3 induced a hypersensitive response in tobacco, whereas RNAi-mediated silencing of PsoRPM3 in transgenic tobacco reduced this hypersensitive response. Several hypersensitive response marker genes were considerably up-regulated in resistant myrobalan plum plants when compared with susceptible counterparts inoculated with M. incognita. PsoPR1a (a SA marker gene), PsoPR2 (a JA marker gene), and PsoACS6 (an ET signaling marker gene) were all more highly expressed in resistant than in susceptible plants. Together, these results support a model in which PsoRPM3 is highly expressed following RKN infection, initiating an HR response that promotes plant resistance through activated salicylic acid, jasmonic acid, and ethylene signaling pathways.

Transcription Factor Pso9TF Assists Xinjiang Wild Myrobalan Plum (Prunus sogdiana) PsoRPM3 Disease Resistance Protein to Resist Meloidogyne incognita.

The root-knot nematode (Meloidogyne incognita) causes huge economic losses in the agricultural industry throughout the world. Control methods against these polyphagous plant endoparasites are sparse, the preferred one being the deployment of plant cultivars or rootstocks bearing resistance genes against Meloidogyne species. Our previous study has cloned one resistance gene, PsoRPM3, from Xinjiang wild myrobalan plum (Prunus sogdiana). However, the function of PsoRPM3 remains elusive. In the present study, we have investigated the regulatory mechanism of PsoRPM3 in plant defense responses to M. incognita. Our results indicate that fewer giant cells were detected in the roots of the PsoRPM3 transgenic tobacco than wild tobacco lines after incubation with M. incognita. Transient transformations of full-length and TN structural domains of PsoRPM3 have induced significant hypersensitive responses (HR), suggesting that TIR domain might be the one which caused HR. Further, yeast two-hybrid results revealed that the full-length and LRR domain of PsoRPM3 could interact with the transcription factor Pso9TF. The addition of Pso9TF increased the ROS levels and induced HR. Thus, our data revealed that the LRR structural domain of PsoRPM3 may be associated with signal transduction. Moreover, we did not find any relative inductions of defense-related genes PsoEDS1, PsoPAD4 and PsoSAG101 in P. sogdiana, which has been incubated with M. incognita. In summary, our work has shown the key functional domain of PsoRPM3 in the regulation of defense responses to M. incognita in P. sogdiana.

4-CPA (4-Chlorophenoxyacetic Acid) Induces the Formation and Development of Defective "Fenghou" (Vitis vinifera × V. labrusca) Grape Seeds.

For some horticultural plants, auxins can not only induce normal fruit setting but also form fake seeds in the induced fruits. This phenomenon is relatively rare, and, so far, the underlying mechanism remains unclear. In this study, "Fenghou" (Vitis vinifera × V. labrusca) grapes were artificially emasculated before flowering and then sprayed with 4-CPA (4-chlorophenoxyacetic acid) to analyze its effect on seed formation. The results show that 4-CPA can induce normal fruit setting in "Fenghou" grapes. Although more seeds were detected in the fruits of the 4-CPA-treated grapevine, most seeds were immature. There was no significant difference in the seed shape; namely, both fruit seeds of the grapevines with and without 4-CPA treatment contained a hard seed coat. However, the immature seeds lacked embryo and endosperm tissue and could not germinate successfully; these were considered defective seeds. Tissue structure observation of defective seeds revealed that a lot of tissue redifferentiation occurred at the top of the ovule, which increased the number of cell layers of the outer integument; some even differentiated into new ovule primordia. The qRT-PCR results demonstrated that 4-CPA application regulated the expression of the genes VvARF2 and VvAP2, which are associated with integument development in "Fenghou" grape ovules. Together, this study evokes the regulatory role of 4-CPA in the division and continuous redifferentiation of integument cells, which eventually develop into defective seeds with thick seed coats in grapes.