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

Abstract

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.

Keywords: Meloidogyne incognita; Pso9TF; PsoRPM3; Xinjiang wild myrobalan plum (Prunus sogdiana); hypersensitive response (HR); tobacco.

Figure 1

Tissue section to observe the disease resistance response of PsoRPM3 transgenic tobacco after nematode inoculation. The paraffin section technique was used to observe the nematode invasion status of tobacco roots infected by M. incognita, and to compare the difference in disease resistance between the transgenic and wild-type tobacco W38. (A–D) The observation of wild-type tobacco W38 roots which were inoculated with M. incognita at 0 d, 3 d, 14 d and 30 d as a negative control; (E–H) The observation of transgenic PsoRPM3 tobacco roots which were inoculated with M. incognita at 0 d, 3 d, 14 d and 30 d; scale bar is 100 μm.

Figure 2

ROS (Reactive oxygen species) burst detection and cell death staining in the PsoRPM3 disease resistance response. (A) Analysis of interactions between PsoRPM3 and PsoRPM3 using the yeast two-hybrid system. α-Galactosidase activity was detected using X-α-Gal as a substrate. (B) Segmentation pattern of the structural domain of PsoRPM3 protein. (C) The brown color is DAB for reactive oxygen species staining in HR reaction, and the blue color is trypan blue for cell necrosis staining. (D) HR responses observed in tobacco (N. benthamiana) leaves transiently overexpressing empty vector, TN, NL, and FL 1 d after inoculation with M. incognita. (E) Determination of cell necrosis area to (D).

Figure 3

Basic characteristics of Pso9TF and detection of interactions with PsoRPM3. (A) Phylogenetic tree containing Pso9TF (rectangle highlighted) and homologous proteins from other related plant species. (B) The predicted three-dimensional structures of the Pso9TF. (C) Pso9TF-GFP localized in the nucleus of N. benthamiana cells, GFP alone localized throughout the whole cells. Fluorescence (left), bright field (middle), and merged images (right) were obtained at 48 h post-agroinfiltration by using Leica confocal microscopy. (D) Transcriptional self-activation of Pso9TF using a yeast one-hybrid. (E) Interaction of Pso9TF with each structural domain of PsoRPM3 using yeast two-hybrid. (F) Interactions between PsoRPM3 and its three chaperonin proteins were determined by BiFC. YFP fluorescence in the upper epidermis cells of N. benthamiana leaves was detected by laser scanning confocal microscopy.

Figure 4

Effect of Pso9TF on PsoRPM3 disease resistance. (A) Pso9TF and GFP empty vectors, Pso9TF and TN, Pso9TF and LRR, Pso9TF and FL were transiently co-expressed on tobacco leaves of N. benthamian, and then injected into nematodes after 1 d, and then stained with DAB for reactive oxygen species. (B) Samples stained with trypan blue for necrotic cells; the left was the test group, the right was the control group. (C) HR reaction observed directly without any dyeing treatment. (D) Measurement and counting of the area of the HR response.

Figure 5

Expression of Pso9TF on downstream disease-resistance related genes. (A–E) Expression of PsoRPM3, Pso9TF, PsoPAD4, PsoEDS1 and PsoSAG101 in the roots of resistant and susceptible Xinjiang wild myrobalan plum (Prunus sogdiana) individuals at 0, 1, 3, 5, and 7 d post-infection. Real-time PCR data were calculated based on three biological and three technical replicates. Error bars = SD.