Increase of Fungal Pathogenicity and Role of Plant Glutamine in Nitrogen-Induced Susceptibility (NIS) To Rice Blast

Abstract

Highlight Modifications in glutamine synthetase OsGS1-2 expression and fungal pathogenicity underlie nitrogen-induced susceptibility to rice blast. Understanding why nitrogen fertilization increase the impact of many plant diseases is of major importance. The interaction between Magnaporthe oryzae and rice was used as a model for analyzing the molecular mechanisms underlying Nitrogen-Induced Susceptibility (NIS). We show that our experimental system in which nitrogen supply strongly affects rice blast susceptibility only slightly affects plant growth. In order to get insights into the mechanisms of NIS, we conducted a dual RNA-seq experiment on rice infected tissues under two nitrogen fertilization regimes. On the one hand, we show that enhanced susceptibility was visible despite an over-induction of defense gene expression by infection under high nitrogen regime. On the other hand, the fungus expressed to high levels effectors and pathogenicity-related genes in plants under high nitrogen regime. We propose that in plants supplied with elevated nitrogen fertilization, the observed enhanced induction of plant defense is over-passed by an increase in the expression of the fungal pathogenicity program, thus leading to enhanced susceptibility. Moreover, some rice genes implicated in nitrogen recycling were highly induced during NIS. We further demonstrate that the OsGS1-2 glutamine synthetase gene enhances plant resistance to M. oryzae and abolishes NIS and pinpoint glutamine as a potential key nutrient during NIS.

Keywords: Magnaporthe oryzae; defense; effector; fertilizer; glutamine; nitrogen; pathogenicity; rice.

Figure 1

Effect of nitrogen supply on rice blast disease in Nipponbare and Kasalath genotypes. Low or high nitrogen fertilization (0N, 1N; see Section Materials and Methods) was applied to rice 1 day before inoculation with different isolates of M. oryzae. The number of susceptible lesions was measured with six different isolates on Nipponbare (A) and Kasalath (B). The mean and sd of three replicates is shown. This experiment was repeated twice and one representative replicate is shown. ^*Student test; P < 0.05; ^***Student test; P < 0.001.

Figure 2

Penetration and growth of M. oryzae in rice plants under different nitrogen regimes. Low or high nitrogen fertilization (0N and 1N; see Section Materials and Methods) was applied to Nipponbare (A) and Kasalath (B) plants which were subsequently inoculated with the Guy11 isolate. At the indicated time after inoculation (1, 2, and 4 dpi), the developmental stage of the fungus was evaluated. Four types of situations were counted: a spore that germinated but did not develop an appressorium (black), a spore with a developed appressorium (dark gray), sites where the fungus had penetrated one cell (light gray) and sites where the fungus had penetrated several cells (white). For each time x treatment combination, a total of 100 events were counted. This experiment was repeated three times and one representative experiment is shown. A Chi square test was used to compare the different percentages (see text). ^***P < 0.001.

Figure 3

Major type of patterns of rice differentially expressed genes during infection under different nitrogen fertilization. (A) Nitrogen specific genes: genes differentially expressed between 0N and 1N treatment but not differentially expressed during infection. (B) Gene expression profile specific of infection by M. oryzae. (C) Gene expression level conserved between 0N and 1N during infection. (D) Enhanced response to inoculation in the 1N condition. (E) Suppression of response to inoculation in 1N condition. (F) Repression by infection only in the 1N condition. Four different conditions were compared with Deseq2 analysis: 0N fertilized and mock inoculated (white), 0N fertilized and Guy11 inoculated (light gray), 1N fertilized and mock inoculated (black dash) and 1N fertilized and Guy11 inoculated (black). The six global patterns indicated here represent the mean and sd expression values of the considered group of genes, as identified by analysis of the p-value obtained for each comparison with Deseq2. For each histogram, a given letter identifies similar values. The values between parentheses represent the number of genes in each category. I, induced; R, repressed; N, nitrogen; Mo, Magnaporthe oryzae; Cons, conserved; En, enhanced; Su, suppressed; Re, repressed. Kasalath plants were fertilized with low (0N) and high (1N) nitrogen fertilization 1 day before inoculation. Plants were inoculated with the Guy11 isolate or a mock solution. RNA were extracted 2 days after infection and analyzed using RNA-seq (see Section Materials and Methods).

Figure 4

Three major pathways potentially in conflict between the host metabolism and pathogen virulence. Adapted from Seifi et al. (2013). Differential expression in RNA-seq at 2 dpi are represented by colored squares for each gene coding for key enzyme. The patterns refer to categories described in Figure 3. The main enzymes implicated in the nitrogen remobilization away from infected tissues are GS, Gln Synthetase; GOGAT, Glutamate synthase; GDC, glycine decarboxylase; ASN, Asn synthetase. The main enzymes implicated in the cell death pathway linked to GS/GOGAT are ProDH, Proline dehydrogenase; ARGAH, P5C reductase, arginase; OAT, Ornithine carbamoyl transferase; NOS, nitric oxide synthase; PAO, polyamine oxidase; GSHS, glutathione synthetase; OsOXO1-4, Oxalate oxidases; OsGLO1, glycolate oxidase. The main enzymes for TCA cycle and GABA-shunt pathways are AST, Asp transaminase; GDH, glutamate dehydrogenase; GAD, glutamate decarboxylase; GABA-T, and SSADH.

Figure 5

Rice blast symptoms in OsGS1-2 mutant and wild type lines. Mutants and corresponding Dongjin WT lines not containing the T-DNA were obtained from the T-DNA insertion line 1B10506 mutated in the OsGS1-2 gene (Only Heterozygous mutants-He- were found; see Supplementary Figure 6). Symptoms caused by M. oryzae (isolate Guy11) were estimated by counting the number of susceptible lesions 5 days after inoculation on eight different replicates. Different doses of nitrogen fertilization (0N, 1N; see Section Materials and Methods) were supplied to rice 1 day before inoculation. This experiment was repeated three times and 12 independent replicates were obtained. Data were normalized between the two replicates using the symptoms obtained for the most susceptible plants in each replicates. ^**Student test; p < 0.01.

Figure 6

Validation of RNA-seq for M. oryzae pathogenicity genes by quantitative RT-PCR. The expression of M. oryzae pathogenicity genes was measured in a biological replicate of the RNA-seq experiment. M. oryzae was inoculated on two varieties (Kasalath and Nipponbare) and only Kasalath showed NIS phenotype as in Figure 1. Gene expression was normalized with fungal actin (MGG_03982). Student test was used to compare 0N and 1N conditions. ^*P < 0.05, ^**P < 0.01. Only the seven significant genes are represented and other genes are shown in Supplementary Figure 8.