Rice ubiquitin-conjugating enzyme OsUbc13 negatively regulates immunity against pathogens by enhancing the activity of OsSnRK1a

Abstract

Ubc13 is required for Lys63-linked polyubiquitination and innate immune responses in mammals, but its functions in plant immunity still remain largely unknown. Here, we used molecular biological, pathological, biochemical, and genetic approaches to evaluate the roles of rice OsUbc13 in response to pathogens. The OsUbc13-RNA interference (RNAi) lines with lesion mimic phenotypes displayed a significant increase in the accumulation of flg22- and chitin-induced reactive oxygen species, and in defence-related genes expression or hormones as well as resistance to Magnaporthe oryzae and Xanthomonas oryzae pv oryzae. Strikingly, OsUbc13 directly interacts with OsSnRK1a, which is the α catalytic subunit of SnRK1 (sucrose non-fermenting-1-related protein kinase-1) and acts as a positive regulator of broad-spectrum disease resistance in rice. In the OsUbc13-RNAi plants, although the protein level of OsSnRK1a did not change, its activity and ABA sensitivity were obviously enhanced, and the K63-linked polyubiquitination was weaker than that of wild-type Dongjin (DJ). Overexpression of the deubiquitinase-encoding gene OsOTUB1.1 produced similar effects with inhibition of OsUbc13 in affecting immunity responses, M. oryzae resistance, OsSnRK1a ubiquitination, and OsSnRK1a activity. Furthermore, re-interfering with OsSnRK1a in one OsUbc13-RNAi line (Ri-3) partially restored its M. oryzae resistance to a level between those of Ri-3 and DJ. Our data demonstrate OsUbc13 negatively regulates immunity against pathogens by enhancing the activity of OsSnRK1a.

Keywords: K63-linked polyubiquitination; OsSnRK1a; OsUbc13; pathogen resistance; rice.

Figure 1

The OsUbc13‐RNAi lines exhibit lesion mimic phenotype accompanied with reactive oxygen species burst and accelerated leaf senescence. (a) qRT‐PCR analysis of OsUbc13 expression in two OsUbc13‐RNAi (Ri‐1 and Ri‐3) lines. OsActin1 gene was used as an internal control. Data are shown as means ±SE; n = 3 (***P < 0.001; Student's t‐test). (b) Whole plants of the wild‐type DJ and two OsUbc13‐RNAi lines at the heading and grain filling stage in the paddy field. Scale bar = 15 cm. (c) Lesion mimic phenotypes and DAB staining on leaves of the OsUbc13‐RNAi plants at 30‐day post‐sowing in soil, compared with that of DJ. Scale bar = 1 cm. (d) Detection of the H₂O₂ content of DJ and the OsUbc13‐RNAi plants at 30‐day post‐sowing in soil. Data are shown as means ±SE; n = 3 (**P < 0.01, ***P < 0.001; Student's t‐test). (e) ROS accumulation dynamics in DJ and OsUbc13‐RNAi plants after flg22 and water (mock) treatments. Data are shown as means ±SE; n = 3. (f) ROS accumulation dynamics in DJ and OsUbc13‐RNAi plants after chitin and water (mock) treatments. Data are shown as means ±SE; n = 3.

Figure 2

The OsUbc13‐RNAi lines display enhanced resistance to both M. oryzae and Xoo. (a) qRT‐PCR analysis of OsUbc13 expression level at different time after inoculation with the compatible isolate GUY11 of M. oryzae. Two‐week‐old wild‐type DJ seedlings were used for inoculation. The seedlings sprayed only with 0.025% Tween 20 were used as negative control (Mock). OsActin1 gene was used as an internal control. Data are shown as means ±SE; n = 3. (b) The lesions on DJ and OsUbc13‐RNAi leaves at 8 days after punch inoculation with the compatible M. oryzae isolate GUY11. Scale bar = 1 cm. (c) Relative lesion area (%) in leaves of (b) indicates significant differences between DJ and OsUbc13‐RNAi. Data are shown as means ±SE; n = 15 (***P < 0.001; Student's t‐test). (d) Relative fungal biomass, measured as MoPot2 by qRT‐PCR, in leaves of (b) was normalized to OsUbq DNA (Park et al., 2012). Data are shown as means ±SE; n = 3 (***P < 0.001; Student's t‐test). (e) The lesions on DJ and OsUbc13‐RNAi leaves at 8 days after spraying inoculation with the compatible M. oryzae isolate GUY11. Scale bar = 1 cm. (f) Relative lesion area (%) in leaves of (e) indicates significant differences between DJ and OsUbc13‐RNAi. Data are shown as means ±SE; n = 5 (***P < 0.001; Student's t‐test). (g) The lesions on DJ and OsUbc13‐RNAi leaves at 18 days after inoculation with the compatible Xoo isolate PXO99. (h) Lesion lengths in leaves of (g) indicate significant differences between DJ and OsUbc13‐RNAi. Data are shown as means ±SE; n = 6 (***P < 0.001; Student's t‐test).

Figure 3

Constitutive expression of several defence‐related genes and contents of defence phytohormones in OsUbc13‐RNAi and DJ. (a) qRT‐PCR was used to analyse the expression of defence‐related genes involved in JA or SA signalling/synthetic pathway. Total RNA was extracted from the leaves of OsUbc13‐RNAi and DJ plants at 30‐day post‐sowing in soil. OsActin1 gene was used as an internal control. Data are shown as means ±SE; n = 3 (**P < 0.01, ***P < 0.001; Student's t‐test). (b) Contents of free salicylic acid (SA), salicylic acid 2‐O‐β‐glucoside (SAG), free jasmonic acid (JA), methyl jasmonate (MEJA), N‐[(−)‐Jasmonoyl]‐(L)‐valine (JA‐Val), and jasmonoyl‐L‐isoleucine (JA‐Ile) in OsUbc13‐RNAi and DJ. The leaves of OsUbc13‐RNAi and DJ plants at 30‐day post‐sowing in soil were used to extract SA, SAG, JA, MEJA, JA‐Val, and JA‐Ile. Data are shown as means ±SE; n = 3 (*P < 0.05, **P < 0.01, ***P < 0.001; Student's t‐test).

Figure 4

OsUbc13 interacts with OsSnRK1a. (a) Y2H assay. The pGBKT7 plasmid containing the OsUbc13 coding sequence (BK‐OsUbc13) and the pGADT7 plasmid containing the OsHRLI, OsYchF1, OsCPI, and OsSnRK1a coding sequence (AD‐OsHRLI, ‐OsYchF1, ‐OsCPI, ‐OsSnRK1a) were co‐transformed into yeast cells (AH109). Yeast cells co‐transformed with AD‐T/BK‐53 or AD‐T/BK‐Lam vectors were used as the positive or negative control, respectively. Interaction of OsUbc13 with OsSnRK1a was indicated by the ability of yeast cells to grow on dropout medium lacking Leu, Trp, His, and Ade for 5 days after plating. (b) LCI assay. Agrobacterial strains containing different combinations of plasmids were co‐infiltrated into tobacco leaves. A cooled charge‐coupled imaging apparatus was used to capture the images. No signal was obtained for the negative controls in which OsSnRK1a‐nLuc was co‐expressed with cLuc, and cLuc‐OsUbc13 was co‐expressed with nLuc. The pseudocolor bar indicates the range of luminescence intensity. (c) Co‐IP assay. OsUbc‐eGFP and OsSnRK1a were co‐expressed in tobacco leaves to detect the interaction between OsUbc13 and OsSnRK1a. An anti‐GFP affinity matrix was used for immunoprecipitation and anti‐OsSnRK1a was used for immunoblot analysis. Red asterisks indicate nonspecific bands. Experiments were repeated two times with similar results. (d) BiFC assay. Fluorescence was observed in the nuclear compartment of transformed tobacco (N. benthamiana) cells, resulting from the complementation of OsSnRK1a‐nYFP+OsUbc13‐cYFP or OsSnRK1a‐cYFP+OsUbc13‐nYFP. No signal was obtained for the negative controls in which OsHRLI‐nYFP was co‐expressed with OsUbc13‐cYFP, and OsHRLI‐cYFP was co‐expressed with OsUbc13‐nYFP. YFP signal was detected by confocal microscopy. Scale bars = 25 μM. (e) Subcellular localization of OsSnRK1a in leaves of tobacco (N. benthamiana). EGFP, enhanced green fluorescent protein; RFP‐H2B, a nuclear marker. Scale bars = 25 μM. (f) Pull‐down assay. 3 × Flag‐OsSnRK1a‐GFP and 8 × His‐OsUbc13‐GFP recombinant proteins expressed by CFPS (cell‐free protein synthesis) reactions were used in the pull‐down assay. Equal amounts of 3 × Flag‐OsSnRK1a‐GFP and 8 × His‐OsUbc13‐GFP were incubated with His‐tag magnetic beads. Pull‐down products were detected using anti‐His and anti‐Flag antibodies. Experiments were repeated two times with similar results.

Figure 5

Y2H screening assay to identify necessary sites and regions required for the interaction between OsUbc13 and OsSnRK1a. (a) Interaction test between OsUbc13 and different truncated OsSnRK1a proteins with specific deletion. Top, the schematic diagram of OsSnRK1a protein. S_TKc, serine/treonine kinase catalytic domain; UBA, ubiquitin associated domain (UBA); KA1, kinase‐associated 1 domain. Low, the interaction was indicated by the ability of yeast cells to grow on dropout medium lacking Leu, Trp, His, and Ade for 5 days after plating. Yeast cells co‐transformed with AD‐T/BK‐53 or AD‐T/BK‐Lam vectors were used as the positive or negative control, respectively. (b) Interaction test between OsUbc13(C89G) and OsSnRK1a, or between OsUbc13 and OsSnRK1a(K43M)/(K139R). Interaction was indicated by the ability of yeast cells to grow on dropout medium lacking Leu, Trp, His, and Ade for 5 days after plating. Yeast cells co‐transformed with AD‐T/BK‐53 or AD‐T/BK‐Lam vectors were used as the positive or negative control, respectively. (c) OsUEV1B interacts with OsSnRK1a. Interaction was indicated by the ability of yeast cells to grow on dropout medium lacking Leu, Trp, His, and Ade for 5 days after plating. Yeast cells co‐transformed with AD‐T/BK‐53 or AD‐T/BK‐Lam vectors were used as the positive or negative control, respectively.

Figure 6

Detection of OsSnRK1a protein content, SnRK1 activity, and polyubiquitination degree. (a) The protein level of OsSnRK1a in leaves of DJ, OsUbc13‐RNAi and ‐OE plants at 30‐day post‐sowing in soil was detected using anti‐OsSnRK1a. Coomassie brilliant blue (CBB) staining was used as a loading control. Band intensity was calculated by ImageJ software. Experiments were repeated three times with similar results. (b) The SnRK1 kinase activity in leaves of DJ and OsUbc13‐RNAi plants at 30‐day post‐sowing in soil. Data are shown as means ±SE; n = 6 (*P < 0.05, **P < 0.01; Student's t‐test). (c) In vivo polyubiquitination level of OsSnRK1a in DJ, OsUbc13‐OE line (OE17‐2), and OsUbc13‐RNAi line (Ri‐3). Crude protein extracted from seedlings at 30‐day post‐sowing in soil was immunoprecipitated by anti‐OsSnRK1a antibody and detected using antibodies that specifically recognize K48/K63‐polyubiquitin conjugates (anti‐K48 and K63) and anti‐OsSnRK1a antibody. Experiments were repeated two times with similar results.

Figure 7

OsOTUB1.1 interacts with OsSnRK1a. (a) Interaction test between OsOTUB1.1 and different truncated OsSnRK1a proteins with specific deletion. Top, the schematic diagram of OsSnRK1a protein. S_TKc, serine/treonine kinase catalytic domain; UBA, ubiquitin‐associated domain; KA1, kinase‐associated 1 domain. Low, the interaction was indicated by the ability of yeast cells to grow on dropout medium lacking Leu, Trp, His, and Ade for 5 days after plating. Yeast cells co‐transformed with AD‐T/BK‐53 or AD‐T/BK‐Lam vectors were used as the positive or negative control, respectively. (b) BiFC assay. Fluorescence was observed in the nuclear compartment of transformed tobacco (N. benthamiana) cells, resulting from the complementation of OsSnRK1a‐nYFP+OsOTUB1.1‐cYFP or OsSnRK1a‐cYFP+OsOTUB1.1‐nYFP. No signal was obtained for the negative controls in which OsSnRK1a^1–455‐nYFP was co‐expressed with OsOTUB1.1‐cYFP, and OsSnRK1a^1–455‐cYFP was co‐expressed with OsOTUB1.1‐nYFP. YFP signal was detected by confocal microscopy. Scale bars = 25 μM. (c) LCI assay. Agrobacterial strains containing different combinations of plasmids were co‐infiltrated into tobacco leaves. A cooled charge‐coupled imaging apparatus was used to capture the images. No signal was obtained for the negative controls in which OsSnRK1a‐nLuc was co‐expressed with cLuc, and cLuc‐OsOTUB1.1 was co‐expressed with nLuc. The pseudocolor bar indicates the range of luminescence intensity.

Figure 8

The OsOTUB1.1‐OE lines display enhanced resistance to M. oryzae. (a) The lesions on ZH11 and OsOTUB1.1‐OE leaves at 8 days after punch inoculation with the compatible M. oryzae isolate GUY11. Scale bar = 1 cm. (b) Relative lesion area (%) in leaves of (a) indicates significant differences between ZH11 and OsOTUB1.1‐OE. Data are shown as means ±SE; n = 12 (***P < 0.001; Student's t‐test). (c) Relative fungal biomass, measured as MoPot2 by qRT‐PCR, in leaves of (a) was normalized to OsUbq DNA (Park et al., 2012). Data are shown as means ± SE; n = 3 (**P < 0.01; Student's t‐test). (d) The protein level of OsSnRK1a in leaves of ZH11 and OsOTUB1.1‐OE plants at 30‐day post‐sowing in soil was detected using anti‐OsSnRK1a. Coomassie brilliant blue (CBB) staining was used as a loading control. Band intensity was calculated by ImageJ software. Experiments were repeated three times with similar results. (e) The SnRK1 kinase activity in leaves of ZH11 and OsOTUB1.1‐OE plants at 30‐day post‐sowing in soil. Data are shown as means ±SE; n = 3 (*P < 0.05; Student's t‐test). (f) In vivo polyubiquitination level of OsSnRK1a in ZH11 and OsOTUB1.1‐OE lines. Crude protein extracted from seedlings at 30‐day post‐sowing in soil was immunoprecipitated by anti‐OsSnRK1a antibody and detected using antibodies that specifically recognize K48/K63‐polyubiquitin conjugates (anti‐K48 and K63) and anti‐OsSnRK1a antibody. Experiments were repeated three times with similar results.

Figure 9

Repression of OsSnRK1a partially reduces the higher resistance to M. oryzae in OsUbc13‐RNAi line (Ri‐3). (a) Expression levels of OsUbc13 and OsSnRK1a in DJ and US‐dRNAi plants. The double RNA interference materials named US‐dRNAi were obtained from the re‐interference of OsSnRK1a in the OsUbc13‐RNAi homozygous line (Ri‐3). OsActin1 gene was used as an internal control. Data are shown as means ±SE; n = 3 (***P < 0.001; Student's t‐test). (b) The protein level of OsSnRK1a in leaves of DJ and US‐dRNAi plants at 30‐day post‐sowing in soil was detected using anti‐OsSnRK1a. Coomassie brilliant blue (CBB) staining was used as a loading control. Experiments were repeated two times with similar results. (c) The lesions on DJ, OsUbc13‐RNAi, and US‐dRNAi leaves at 8 days after punch inoculation with the compatible M. oryzae isolate GUY11. Scale bar = 1 cm. (d) Relative lesion area (%) in leaves of (b) indicates significant differences between DJ, OsUbc13‐RNAi, and US‐dRNAi. Data are shown as means ±SE; n = 9. Significant difference was determined by ANOVA; values with different letters indicate a significant difference from each other (P < 0.05). (e) Relative fungal biomass, measured as MoPot2 by qRT‐PCR, in leaves of (b) was normalized to OsUbq DNA (reference). Data are shown as means ±SE; n = 3 (**P < 0.01; Student's t‐test).

Figure 10

A proposed model for OsUbc13‐mediated blast resistance. In wild‐type DJ plants, OsUbc13 interacts with OsSnRK1a, resulting in high levels of K63‐linked polyubiquitination on OsSnRK1a, inhibition of its activity, and becoming susceptible to M. oryzae. In the OsUbc13‐RNAi plants, a small amount of OsUbc13 does not allow more K63‐linked polyubiquitination on OsSnRK1a, leading to increased activity of OsSnRK1a and enhanced resistance to M. oryzae.