Quantitative Proteomic Analysis Provides Insights into Rice Defense Mechanisms against Magnaporthe oryzae

Abstract

Blast disease is one of the major rice diseases, and causes nearly 30% annual yield loss worldwide. Resistance genes that have been cloned, however, are effective only against specific strains. In cultivation practice, broad-spectrum resistance to various strains is highly valuable, and requires researchers to investigate the basal defense responses that are effective for diverse types of pathogens. In this study, we took a quantitative proteomic approach and identified 634 rice proteins responsive to infections by both Magnaporthe oryzae strains Guy11 and JS153. These two strains have distinct pathogenesis mechanisms. Therefore, the common responding proteins represent conserved basal defense to a broad spectrum of blast pathogens. Gene ontology analysis indicates that the “responding to stimulus” biological process is explicitly enriched, among which the proteins responding to oxidative stress and biotic stress are the most prominent. These analyses led to the discoveries of OsPRX59 and OsPRX62 that are robust callose inducers, and OsHSP81 that is capable of inducing both ROS production and callose deposition. The identified rice proteins and biological processes may represent a conserved rice innate immune machinery that is of great value for breeding broad-spectrum resistant rice in the future.

Keywords: Magnaporthe oryzae; basal defense; iTRAQ; innate immunity; proteomics; rice blast.

Figure 1

Gene ontology (GO) analysis on differentially expressed (DE)-proteins and their distribution map. (A) Venn diagram shows the expression preference of the 634 DE-proteins; (B) Gene ontology analysis of the 634 DE-proteins. The pie chart indicates the biological process of 634 DE-proteins. The fan-shaped chart shows the subterms of the 40 DE-proteins responding to stimulus.

Figure 2

Biological processes (BP) analysis on DE-proteins and their expression preference. The 634 DE-proteins were analyzed by using the DAVID Bioinformatics Resources 6.8. Biological processes were sorted according to their enrichment in both Guy11 and JS153 infections. The terms in italic indicate sub-terms that belong to the major terms above.

Figure 3

Expression coalition between the transcription and the translation levels. Top: expression of the six selected genes examined by qRT-PCR at the indicated time points by Guy11 infection or JS153 infection. Values represent the means ± SD of three independent samples (* p < 0.05, ** p < 0.01). Similar results were obtained from three biological repeats. Bottom: protein abundance of the six selected genes. Error bars indicated SD. Asterisks indicate significant differences (* p <0.05, ** p < 0.01).

Figure 4

The DEP candidates contribute to callose deposition and ROS accumulation. (A) Left: 3,3′-diaminobenzidine (DAB) staining for reactive oxygen species (ROS) accumulation in N. benthamiana leaves after infiltration with A. tumefaciens carrying empty vector, PcINF1, and the six selected genes. The reddish-brown at the injection site shows the accumulation of ROS. Numbers are the relative accumulation and standard deviations of ROS using Image J. Right: Histogram represents the relative accumulation of ROS in the images. Error bars indicate SD from three technical replicates. Asterisks indicate significant differences (* p < 0.05, ** p < 0.01); (B) Left: Aniline blue staining for callose deposition in the leaves (40× magnification) expressing EV (empty vector), PsNLP and six selected genes. Numbers are the means and standard deviations of three 1 cm² microscopic fields of view. Right: Histogram represents the means of three 1 cm² microscopic fields of view. Error bars indicate SD. Asterisks indicate significant differences (** p < 0.01).

Figure 5

The defense signaling pathway in response to M. oryzae in rice. Critical components involved in the SA, JA, and ET signaling pathways are analyzed by real-time PCR. Rectangles indicate genes or proteins; ovals indicate chemical compounds, red ovals indicate phytohormones. The chart with different colors indicates the expression of indicated genes at the indicated time points by Guy11 infection or JS153 infection.