OsPGIP1-Mediated Resistance to Bacterial Leaf Streak in Rice is Beyond Responsive to the Polygalacturonase of Xanthomonas oryzae pv. oryzicola

Abstract

Polygalacturonase-inhibiting proteins (PGIPs) have been shown to recognize fungal polygalacturonases (PGs), which initiate innate immunity in various plant species. Notably, the connection between rice OsPGIPs and PGs in Xanthomonas oryzae pv. oryzicola (Xoc), which causes bacterial leaf streak (BLS), remains unclear. Here, we show that OsPGIP1 was strongly induced after inoculating rice with the Xoc strain RS105. Furthermore, OsPGIP1-overexpressing (OV) and RNA interference (RNAi) rice lines increased and decreased, respectively, the resistance of rice to RS105, indicating that OsPGIP1 contributes to BLS resistance. Subsequently, we generated the unique PG mutant RS105Δpg, the virulence of which is attenuated compared to that of RS105. Surprisingly, the lesion lengths caused by RS105Δpg were similar to those caused by RS105 in the OV lines compared with wild-type ZH11 with reduced Xoc susceptibility. However, the lesion lengths caused by RS105Δpg were still significantly shorter in the OV lines than in ZH11, implying that OsPGIP1-mediated BLS resistance could respond to other virulence factors in addition to PGs. To explore the OsPGIP1-mediated resistance, RNA-seq analysis were performed and showed that many plant cell wall-associated genes and several MYB transcription factor genes were specifically expressed or more highly induced in the OV lines compared to ZH11 postinoculation with RS105. Consistent with the expression of the differentially expressed genes, the OV plants accumulated a higher content of jasmonic acid (JA) than ZH11 postinoculation with RS105, suggesting that the OsPGIP1-mediated resistance to BLS is mainly dependent on the plant cell wall-associated immunity and the JA signaling pathway.

Keywords: Bacterial leaf streak; Cell wall-associated genes; Defense; Jasmonic acid; Polygalacturonase-inhibiting proteins; Rice; Sheath blight.

Fig. 1

Expression patterns of rice OsPGIPs in response to the Xanthomonas oryzae pv. oryzicola strain RS105. a Relative expression of OsPGIPs in ZH11 in response to Xoc. The gene expression levels of OsPGIP1 to OsPGIP7 were analyzed by qRT-PCR after inoculation with Xoc stain RS105 at 2, 4, 8, 24, and 96 h. b The expression of OsPGIP1 in response to RS105 at 6, 24, 48, and 96 h in the susceptible rice variety ZH11 and moderately resistant variety Acc8558. The housekeeping gene ACTIN was used to normalize the data. Error bars represent the standard deviations for three replicates. Three independent experiments were performed with the same expression pattern

Fig. 2

OsPGIP1 contributes to bacterial leaf streak resistance. a The phenotypes of the RS105 lesions that developed on ZH11 and two OsPGIP1-overexpressing lines (OV-12 and OV-24) at 14 days after infiltration. The arrows represent the boundaries of lesion expansion caused by RS105 infection. b Relative expression fold change of OsPGIP1 in OV-12 and OV-24. The expression of wild-type ZH11 was used as a control and set to 1. The housekeeping gene ACTIN was used to normalize the data. c The lesion lengths of ZH11, OV-12 and OV-24 at 14 days after inoculation with RS105. d The phenotypes of the RS105 lesions that developed on ZH11 and two OsPGIP1-suppressed lines (RNAi-7 and RNAi-10) at 14 days after infiltration. The arrows represent the boundaries of lesion expansion caused by RS105 infection. e Relative expression level of OsPGIP1 in RNAi-7, RNAi-10 and ZH11. The expression of wild-type ZH11 was used as a control and set to 1. ACTIN was an internal reference gene for normalization. f The lesion lengths of ZH11, RNAi-7 and RNAi-10 inoculated with RS105 after 14 days. Data were analyzed using a t-test. Asterisks represent statistically significant differences from the ZH11 wild type at P < 0.05

Fig. 3

The polygalacturonase gene acts as a virulence factor of RS105 during infection in rice. a The position of the polygalacturonase (PG) gene in the Xoc RS105 genome. b The phenotype of RS105 and the PG mutant strain (RS105Δpg) on the susceptible rice variety ZH11 and moderately resistant variety Acc8558. Images were photographed 14 days after inoculation. c The statistical counting of lesion length at 14 days after inoculation with RS105 and RS105Δpg on ZH11 and Acc8558. The data were counted from over 10 plants and analyzed using a t-test (P < 0.05). Asterisks represent statistically significant differences from ZH11. d The bacterial growth curves of RS105 and RS105Δpg in ZH11 and Acc8558 rice at 1, 4, and 7 days. Significant differences were determined by t test: *P < 0.05. e Lesion length after inoculation with RS105, RS105Δpg and PG gene complementary stain (RS105Δpg-CP) at 14 days in ZH11. The letters above the bars represent the significant differences at a value of P ≤ 0.05 (LSD test). The above experiments were repeated three times with similar results

Fig. 4

OsPGIP1-mediated resistance is complemented and induced by XocPG in addition to other Xoc pathogenicity factors. Lesion lengths of RS105, RS105Δpg and RS105Δpg-CP in ZH11 and OsPGIP1-overexpressing lines OV-12 and OV-24. The letters above the bars represent the significant differences at a value of P ≤ 0.05 (LSD test). The above experiment was repeated three times with similar results

Fig. 5

RNA-seq analysis of differentially expressed genes between the OsPGIP1-overexpressing line (OV-24) and ZH11 in response to RS105 inoculation. a and b Venn diagram of upregulated (a) or downregulated (b) DEGs in response to RS105 at 24 h postinoculation. DEGs were considered at P value< 0.001 and |log2-fold change| > 1. ZH11-RS vs. ZH11 and OV-24-RS vs. OV-24 represent the common DEGs identified in two experimental repeats, with noninfected ZH11 and OV-24 as controls. c Heatmap analysis of the common significantly upregulated DEGs in OV-24-RS vs. OV-24 and ZH11-RS vs. ZH11. The heatmap was classified into defense response, cell wall metabolism, transcription factors (TFs), receptor-like kinases (RLKs) and ROS metabolism. The intensity of color indicates the expression level of genes from left to right, indicating higher gene expression. d The expression of the transcription factor genes, cell wall-related genes and defense response genes that were upregulated in RNA-seq analysis was evaluated by qRT-PCR in ZH11 and OV-24. The internal control gene was ACTIN. The above experiments were repeated three times with similar results

Fig. 6

Increased accumulation of jasmonic acid in OsPGIP1-overexpressing compared with ZH11 after inoculation with Xoc. The SA and JA contents were measured in the ZH11 and OsPGIP1-overexpressing lines OV-12 and OV-24 without inoculation and 24 h postinoculation with RS105. The letters above the bars represent the significant differences at a value of P ≤ 0.05 (LSD test)