RESISTANCE TO POWDERY MILDEW8.1 boosts pattern-triggered immunity against multiple pathogens in Arabidopsis and rice

Abstract

The Arabidopsis gene RESISTANCE TO POWDERY MILDEW8.1 (RPW8.1) confers resistance to virulent fungal and oomycete pathogens that cause powdery mildew and downy mildew, respectively. However, the underlying mechanism remains unclear. Here, we show that ectopic expression of RPW8.1 boosts pattern-triggered immunity (PTI) resulting in enhanced resistance against different pathogens in both Arabidopsis and rice. In Arabidopsis, transcriptome analysis revealed that ectopic expression of RPW8.1-YFP constitutively up-regulates expression of many pathogen-associated molecular pattern (PAMP-)-inducible genes. Consistently, upon PAMP application, the transgenic line expressing RPW8.1-YFP exhibited more pronounced PTI responses such as callose deposition, production of reactive oxygen species, expression of defence-related genes and hypersensitive response-like cell death. Accordingly, the growth of a virulent bacterial pathogen was significantly inhibited in the transgenic lines expressing RPW8.1-YFP. Conversely, impairment of the PTI signalling pathway from PAMP cognition to the immediate downstream relay of phosphorylation abolished or significantly compromised RPW8.1-boosted PTI responses. In rice, heterologous expression of RPW8.1-YFP also led to enhanced resistance to the blast fungus Pyricularia oryzae (syn. Magnaporthe oryzae) and the bacterial pathogen Xanthomonas oryzae pv. oryzae (Xoo). Taken together, our data suggest a surprising mechanistic connection between RPW8.1 function and PTI, and demonstrate the potential of RPW8.1 as a transgene for engineering disease resistance across wide taxonomic lineages of plants.

Keywords: Pseudomonas syringae; Pyricularia oryzae; RPW8; Xanthomonas oryzae; disease resistance; pattern-triggered immunity.

Figure 1

Ectopic expression of RPW8.1‐YFP enhances resistance to bacterial pathogens in Arabidopsis. (a‐d) Bacterial growth assay for the indicated strains in the indicated transgenic lines in comparison with the wild‐type (WT) Col‐gl. Error bars indicate standard deviation (SD, n = 6). Different letters above the bars indicate significant differences (P < 0.01) as determined by a one‐way ANOVA followed by post hoc Tukey HSD analysis. Similar results were obtained in three independent experiments.

Figure 2

Ectopic expression of RPW8.1‐YFP constitutively up‐regulates the expression of PAMP‐inducible genes in Arabidopsis. (a) Comparison of gene numbers between PAMP‐inducible genes in wild‐type (WT) Col‐gl and constitutively up‐regulated genes in R1Y4. (b, c) Quantitative RT‐PCR data show that relative mRNA levels of the indicated genes upon application of flg22 (b) and chitin (c) at the indicated time points. Error bars indicate SD (n = 3). Student's t‐test was carried out to determine the significance of difference between WT and R1Y4 at 0 h postinfiltration (HPI). Asterisks (**) indicated significant difference at P ≤ 0.01. Similar results were obtained in two independent experiments.

Figure 3

Ectopic expression of RPW8.1‐YFP results in enhanced PAMP‐induced defence responses. (a) Western blot analysis shows MAPK activation in wild‐type (WT) Col‐gl, R1Y4 and R2Y4 by flg22 at the indicated time points. Phosphorylated MAPKs were detected by anti‐pERK sera. Ponceau S‐stained rubisco was used as loading control. (b, c) Comparison of PAMP‐induced burst of reactive oxygen species (ROS) in WT, R1Y4 and R2Y4. Error bars indicate SD (n = 4). (d) Comparison of PAMP‐induced callose deposition in WT, R1Y4 and R2Y4. (e) Quantitative RT‐PCR data show the expression pattern of the indicated PTI marker genes in WT, R1Y4 and R2Y4 upon application of flg22 or chitin. Relative mRNA level was normalized to that in WT at 0 hr. Error bars indicate SD (n = 3). Different letters above the bars indicate significant differences at P < 0.01. All the experiments were repeated two times with similar results.

Figure 4

Ectopic expression of RPW8.1‐YFP results in PAMP‐induced ETI‐like responses. (a) DAB ‐(3,3’‐diaminobenzidine‐) and trypan blue‐stained leaves from the indicated lines show H₂O₂ production and dead cells, respectively. (b‐e) Micrographs show flg22/chitin‐induced ETI‐like responses in R1Y4. Note that during early period of PAMP treatment, H₂O₂ was enriched in cells, especially in chloroplasts, and the cells were intact (b). Then, the H₂O₂‐accumulated cells were shrunken in the apoplastic space along the neighbouring cells, but the chloroplasts (arrows) were still visible (c). Finally, bubbles (arrows) from the shrinking cytoplasm (arrowheads) were formed in some neighbouring cells of the shrunken cells (*) (d), and eventually formed clusters of dead cells as demonstrated by trypan blue staining (e). Size bar, 10 μm. (f) Micrographs show flg22‐/chitin‐induced H₂O₂ in WT and R2Y4 cells. Note that flg22‐ and chitin‐induced H₂O₂ accumulation and cell death were not observed in WT cells, but occasionally observed in R2Y4 cells. (g, h) Expression pattern of defence‐related genes PR1 and PR2 in the indicated lines upon application of flg22 (g) and chitin (h). Relative mRNA levels were normalized to that in WT at 0 hr. Error bars indicate SD (n = 3). Different letters above the bars indicate significant differences at P < 0.01. Similar results were obtained in two independent experiments.

Figure 5

Ectopic expression of RPW8.1‐YFP activates multiple layers of defence responses to a virulent bacterial pathogen. (a, c) Quantitative RT‐PCR analysis on the expression of the indicated marker genes upon infection of P. syringae DC3000. Error bars indicate SD (n = 3). Different letters above the bars indicate significant differences at P < 0.01. Similar results were obtained in two independent experiments. (b, d) P. syringae DC3000 induced callose deposition (b), H₂O₂ production and cell death (d) in the indicated lines revealed by aniline blue, DAB and trypan blue staining, respectively. (e) Adenylate cyclase activity assay shows the different capability of effector secretion in WT and R1Y4 upon P. syringae DC3000 infection. The cAMP accumulation was normalized to the bacterial colony numbers at the indicated hours after infiltration (HPI). (f, g) Comparison of AvrPto‐ or HopAi1‐mediated suppression on flg22‐induced FRK1::LUC expression. The LUC reporter activity (%) was normalized to the activity of empty vector. Values were normalized to the internal control 35S::RLUC . Error bars indicate SD (n = 3). Different letters above the bars indicate significant differences at P < 0.01. Similar results were obtained in three independent experiments.

Figure 6

PTI signalling is required for RPW8.1‐enhanced defence responses. (a, b) Burst of reactive oxidative species (ROS) induced by flg22 (a) and chitin (b) in the indicated lines in comparison with the wild‐type (WT) Col‐gl. Error bars indicate SD (n = 4). (c) Quantitative analysis of PAMP‐induced callose deposition in the indicated lines. Error bars indicate standard deviation (SD, n = 6). (d, e) Bacterial growth assay for P. syringae DC3000(hrcC ^‐ ) (d) and P. syringae DC3000 (e) in the indicated lines. Error bars indicate SD (n = 4). (f) P. syringae DC3000‐induced callose deposition in the indicated lines. Error bars indicate SD (n = 6). (g) Comparison of AvrPto‐ or HopAi1‐mediated suppression on flg22‐induced FRK1::LUC expression in the indicated lines. The PFRK1:LUC reporter activity (%) was normalized to the activity in WT protoplasts transfected without effectors. Error bars indicate SD (n = 3). Different letters above the bars in (c‐g) indicate significant differences at P < 0.01. All the experiments were independently repeated twice with similar results.

Figure 7

Ectopic expression of RPW8.1‐YFP enhances resistance against P. oryzae in rice. (a‐d) Representative leaf sections from the indicated transgenic lines and the wild‐type (WT) TP309 show the blast disease phenotypes (a, b) and statistical analyses on the lesion area (c, d) caused by the P. oryzae strain Guy11 (a, c) and the eGFP‐tagged strain GZ8 (b, d). Error bars indicate SD (n = 10). (e) P. oryzae induced expression of the indicated defence‐related genes in the indicated transgenic rice lines in comparison with WT. Relative mRNA levels were normalized to that in untreated WT plants. Error bars indicate SD (n = 3). Different letters above the bars in (c‐e) indicate significant differences at P < 0.01. All the experiments were independently repeated two times with similar results.

Figure 8

Ectopic expression of RPW8.1‐YFP in rice enhances postinvasive resistance to P. oryzae. (a) Confocal images show the infection process of the eGFP‐tagged strain Zhong‐8‐10‐14 on sheath cells from the wild‐type (WT) TP309 and the transgenic line expressing RPW8.1‐YFP . Note that appressoria (arrows) were observed at 12 h postinoculation (HPI). Invasive hyphae (red arrowheads) in the primary infected cells were observed at 24 HPI. The invasive hyphae extended to the neighbour cells (white arrowheads) at 36 HPI. Size bars, 20 μm. Similar results were obtained in two independent experiments. (b) Quantitative analyses on the process of infection from at least 50 conidia at the indicated time points on the indicated lines. Note that the number of invasive hyphae extended from the primary infected cells into the neighbouring cells is obviously lower in the RPW8.1‐YFP transgenic line than that in the control WT at 36 HPI (*).

Figure 9

Ectopic expression of RPW8.1‐YFP in rice enhances resistance to Xoo. (a) Leaf sections from the indicated transgenic lines show the disease phenotypes in comparison with the wild‐type (WT) TP309 at 14 days postinoculation (dpi) of Xoo. Similar results were obtained in two independent experiments. (b, c) Statistical analysis on lesion length (b) and bacterial growth (c) at 14 dpi. Error bars indicate SD (n = 10). Different letters above the bars indicate significant differences at P < 0.01. All the experiments were independently repeated twice with similar results. (d) A hypothetical model for the RPW8.1‐PTI connection.