OsWRKY IIa Transcription Factors Modulate Rice Innate Immunity
© The Author(s) 2010
Received: 17 November 2009
Accepted: 3 February 2010
Published: 20 February 2010
WRKY transcription factors regulate diverse plant processes including responses to biotic stresses. Our previous studies indicate that OsWRKY62, an OsWRKY IIa subfamily member, functions as a negative regulator of the rice defense against Xanthomonas oryzae pv. oryzae. Here, we report that a large inverted repeat construct designed to knock down the expression of the four OsWRKY IIa subfamily members (OsWRKY62, OsWRKY28, OsWRKY71, and OsWRKY76) leads to overexpression of all four genes and disease resistance in some transgenic plants. These phenotypes are stably inherited as reflected by progeny analysis. A pathogenesis-related gene, PR10, is up-regulated in plants overexpressing the OsWRKY IIa genes. These results suggest that OsWRKY IIa proteins interact functionally to modulate plant innate immunity.
WRKY proteins constitute a family of mostly plant-specific zinc-finger transcription factors composed of 74 and 109 members in Arabidopsis and rice, respectively (Eulgem and Somssich 2007; Ross et al. 2007). A common feature of WRKY proteins is the WRKY domain, which is defined by a region of approximately 60 amino acids containing a conserved WRKYGQK amino acid sequence adjacent to a zinc-finger-like motif (Eulgem et al. 2000). Both regions are necessary for high-affinity binding to a canonical W-box motif containing a consensus sequence (C/T)TGAC(C/T). Based on primary structure, WRKY proteins have been classified into three groups (I, II, and III) and various subgroups (e.g., IIa, IIb, etc.).
Recent studies have shown that WRKY transcription factors are involved in various biological processes but most notably respond to biotic and abiotic stresses (Pandey and Somssich 2009). WRKY transcription factors from various plant species function as both positive and negative regulators in plant innate immunity. Compared with the gain-of-function analysis which has been a powerful tool to reveal defense-related phenotype of individual WRKY transcription factor, the success of loss-of-function analysis has been limited due to functional redundancy among structurally related WRKY proteins (Wang et al. 2006; Xu et al. 2006). For example, Piffanelli and colleagues screened 20 Oswrky insertion mutants but observed no phenotypic change for host and non-host pathogen interaction (Berri et al. 2009). Only a few WRKY genes have been reported to show an altered defense response in knockout or knock-down plants. For example, loss of AtWRKY70 function compromises both basal defense response to bacterial and fungal pathogens and RPP4-mediated race-specific resistance to Hyaloperonospora parasitica (Li et al. 2004; Li et al. 2006a; Knoth et al. 2007). Knock-down of AtWRKY23 expression results in decreased susceptibility to the cyst nematode Heterodera schachtii (Grunewald et al. 2008). OsWRKY89 suppression lines show reduced wax content and increased susceptibility to rice blast fungus (Wang et al. 2007).
To study the function of WRKY family genes with functional redundancies, one strategy is to generate lines in which multiple WRKY genes are knocked-out. For example, knock-outs of AtWRKY IIa subfamily genes AtWRKY18, AtWRKY40 and AtWRKY60 individually does not affect the plant response to the hemibiotrophic bacterial pathogen Pseudomonas syringae and the necrotrophic fungal pathogen Botrytis cinerea. However, wrky18 wrky40 and wrky18 wrky60 double mutants and the wrky18 wrky40 wrky60 triple mutant are more resistant to P. syringae but more susceptible to B. cinerea (Xu et al. 2006). The Arabidopsis WRKY IId subfamily member wrky11 but not wrky7 or wrky17 mutants show enhanced basal resistance P. syringae. Further enhanced resistance is observed in wrky11 wrky17 double mutant plants that are generated and tested (Journot-Catalino et al. 2006).
We have previously reported that overexpression of OsWRKY62 compromises resistance to Xanthomonas oryzae pv. oryzae (Xoo) in the presence and absence of the Xa21 pattern recognition receptor (Peng et al. 2008; Lee et al. 2009). OsWRKY62, together with OsWRKY28, OsWRKY71 and OsWRKY76, comprise the rice WRKY IIa subfamily. It has been shown that transcripts of OsWRKY62 and OsWRKY76 accumulate in response to benzothiadiazole, salicylic acid (SA) and the rice fungal pathogen Magnaporthe grisea (Ryu et al. 2006; Shimono et al. 2007). In addition, OsWRKY71 is induced by SA and rice bacterial pathogen treatment (Liu et al. 2007). To understand the complex of this phylogenetically related gene family in the rice innate immunity, we designed a large inverted repeat construct to knock-down expression of all four members. Intriguingly, this silencing construct led to over-expression of all four genes in some transgenic plants. Those lines showing this “super-overexpression” phenotype displayed resistance against Xoo. In these super-overexpression resistant lines, pathogenesis-related (PR) gene expression was activated. Although the mechanism of the super-overexpression phenotype induced by the gene silencing construct is unknown, our results indicate functional interactions between OsWRKY IIa subfamily members in regulating plant innate immunity.
Generation of a super long inverted repeat construct
To probe the role of the OsWRKY IIa subfamily genes in plant innate immunity, we attempted to knock down all four family members simultaneously with one “super” long inverted repeat (SIR) construct. SIR constructs have been used to successfully silence various numbers of a family of four Rac GTPases (Miki et al. 2005).
Endogenous OsWRKY IIa gene expression in the WRKYssRNAi transgenic lines
Super-overexpression of OsWRKY IIa subfamily genes enhances rice basal defense against Xoo
To elucidate the molecular basis of the disease resistance phenotype developed on plants super-overexpressing OsWRKY IIa genes, we carried out RT-PCR analysis to compare the expression of PR10, a defense-related gene, in un-inoculated leaves. As seen in Fig. 2a, PR10 was highly induced in the lines super-overexpressing OsWRKY IIa members when compared to the Kitaake controls. These results indicate that super-overexpression of OsWRKY IIa genes activates pathogenesis-related gene expression, which likely contributes to enhanced basal defense against Xoo in rice plants.
The disease resistance and super-overexpression phenotypes are stably inherited
To verify that the disease resistance phenotype correlated with super-overexpression of OsWRKY IIa genes, we analyzed the expression of OsWRKY IIa genes in the T2 progeny from WRKYssRNAi T1 lines, 11-2, -26-6, and -30-1. RNA samples were extracted from un-inoculated leaves. RT-PCR results in Fig. 4d shows that the progeny that inherited the WRKYssRNAi construct maintained the elevated expression level of OsWRKY IIa genes. Together, these results indicate that super-overexpression of OsWRKY IIa genes caused by the WRKYssRNAi construct can by stably inherited. Moreover, super-overexpression of OsWRKY IIa genes enhances resistance to Xoo in rice.
OsWRKY IIa subfamily members play both positive and negative regulatory roles in regulating plant innate immunity. Overexpression of OsWRKY62 compromises rice basal defense and Xa21-mediated resistance to Xoo (Peng et al. 2008). By contrast, overexpression of OsWRKY71 results in elevated expression of defense-related genes and enhanced resistance to Xoo (Liu et al. 2007). The work presented here demonstrates that super-overexpression of OsWRKY IIa subfamily members enhances basal defense against Xoo. These data indicate functional interactions between OsWRKY IIa proteins in regulating plant innate immunity. Yeast two-hybrid and bimolecular fluorescence complementation experiments provide support for physical interactions among the OsWRKYIIa proteins (Y. Seo et al., in preparation).
Similarly, Arabidopsis WRKY IIa subfamily members interact both physically and functionally in plant defense (Xu et al. 2006). AtWRKY IIa proteins form both homocomplexes and heterocomplexes. Association of different WRKY IIa members can alter their sequence-specific DNA binding activities. In addition, constitutive expression of AtWRKY18 enhances resistance to P. syringae, while plants overexpressing AtWRKY18 with either AtWRKY40 or AtWRKY60 are more susceptible to both P. syringae and B. cinerea. These observations indicate that association of different WRKY IIa members may alter defense gene regulation and ultimately affect the plant defense response.
A recent report on interactions of two transcriptional repressors and two transcriptional activators in modulating gibberellin signaling in aleurone cells provides an intriguing example of how different transcription factor complex affects the expression of target genes (Zou et al. 2008). HvWRKY38, a barley ortholog of OsWRKY71, functions as a repressor of GA-induced expression of Amy32b a-amylase. When present individually, the inductive activities of SAD, a DNA-binding with one finger (DOF) protein and HvGAMYB (a R2R3 MYB protein) on Amy32b expression are blocked by HvWRKY38. However, SAD and HvGAMYB together overcome the inhibitory effect of HvWRKY38. Interestingly, the synergistic effect of SAD and HvGAMYB transcriptional activators is almost abolished when repressor BPBF, a barley prolamin-box binding factor which is another DOF protein, is present with HvWRKY38. Further investigation shows that HvWRKY38 interacts with HvBPBF in the nuclei of aleurone cells. Moreover, HvWRKY38 interferes with the binding of HvGAMYB to the Amy32b promoter. Taken together, these studies suggest that relative amounts of activator and repressor proteins in a complex can modulate the expression of target genes.
OsWRKY IIa proteins contain putative leucine zipper motifs at the N-terminus, which suggests potential homodimerization or heterodimerization between proteins. It has been shown that leucine zipper motifs are critical for the physical interaction of AtWRKY IIa proteins and homodimerization of HvWRKY38 (Xu et al. 2006; Zou et al. 2008). OsWRKY71 has also recently been reported to function as a transcriptional repressor (Chujo et al. 2008). As a positive regulator of plant defense response, OsWRKY71 may play an indirect role in transcriptional activation of defense-related gene expression by interacting with other regulatory proteins. Taken together, we hypothesize that different combinatorial dimers formed by OsWRKY IIa proteins may exhibit different functions in regulating target gene expression. Moreover, changes in the participants within protein complexes caused by mutation, overexpression or environmental stress can also affect the expression of target genes, leading to altered plant defense response. This may explain the data observed with rice lines overexpressing OsWRKY62 alone (susceptible) compared with plants overexpressing all four OsWRKY IIa proteins (resistant).
While ineffective gene silencing by short interference RNA (siRNA) is commonly observed (Rose et al. 2005), it has not previously been observed that a siRNA construct can lead to super-overexpression of the target genes. Inverted repeats are usually found to be very efficient for silencing (Miki et al. 2005), although different levels of efficiency of silencing are not uncommon (Chern et al. 2005a). We have searched the literature for an explanation of the super-overexpression phenomenon and found reports of RNAs that are able to stimulate transcription; however, these examples are directed toward promoter sequences, not the translated region of a gene. For instance, Dahiya and associates discovered that exogenous dsRNAs designed against human promoter regions increase expression of the corresponding coding region (Li et al. 2006b). Our results indicate that simultaneous over-expression of all the members of the rice WRKY IIa complex reveals a previously unobserved outcome of attempted gene silencing. Although the mechanism remains unknown at this point, one possibility is that self-regulation by WRKY genes or regulation via other transcription factors and proteins may contribute to this phenotype.
Plant materials and growth conditions
Rice (Oryza sativa L.) plants were grown in the greenhouse until 5 weeks of age and transferred to the growth chamber before Xoo inoculation. In the greenhouse, the light intensity in photosynthetic photon flux across the spectrum from 400 to 700 nm was approximately 250 μmol m−2 s−1 in spring. The growth chamber was set on a 14-hour daytime period, a 28/26°C temperature cycle and at 90% humidity. The chamber was equipped with metal halide and incandescent lights. The light intensity in the growth chamber was approximately 100 μmol m−2 s−1.
Xoo inoculation and quantification of bacterial populations
For Xoo inoculation, a bacterial suspension (OD600 of 0.5) of Xoo stain PX099 (Philippine race 6, PR6), which carries ax21 activity, was used to inoculate rice by the scissors-dip method (Kauffman et al. 1973; Lee et al. 2009).
To determine the bacterial populations, three inoculated leaves from each genotype were ground up and resuspended in 10 mL H2O to harvest bacteria separately. Diluted extract was plated on peptone sucrose agar plates containing 15 mg/L cephalexin.
Plasmid construction and rice transformation
To generate the WRKYssRNAi construct, the fragments of OsWRKY62 and OsWRKY71 were fused by overlapping PCR using primers 5′-TCGCCGACCAAGGATTGAGCACGATCACACTCGAC-3′/5′-TTTGGATCCCAGGAATGTGTGGGAT-3′ (for OsWRKY62); 5′-TCCAAGCGCTTCGTCCAGAACAGCGACGGCTCCG-3′/5′-GTCGAGTGTGATCGTGCTCAATCCTTGGTCGGC-3′ (for OsWRKY71); The fragments of OsWRKY28 and OsWRKY76 were fused by overlapping PCR using primers 5′-TTTGAATTCCTGCGACGCCAA-3′/5′-GAGGCTGTCGACGCTAATTGGTACCCGTCCTTGA-3′ (for OsWRKY76); 5′-TCAAGGACGGGTACCAATTAGCGTCGACAGCCTCGA-3′/5′-CGGAGCCGTCGCTGTTCTGGACGAAGCGCTTGGAG-3′ (for OsWRKY28). Both PCR products were purified and fused by overlapping PCR using primers 5′-TTTGATTCCTGCCGACGCCAA-3′/5′-TTTGGATCCCAGGAATGTGTGGGAT-3′. The final product was cloned into the pGEM T Easy vector (Promega) and confirmed by sequencing. The insert was then excised with BamHI and EcoRI and ligated with a 1 kb, EcoRI-digested GUS fragment into BamHI pre-cut pBlueScript II SK-, yielding a 2.6 kb insert containing fragments from four WRKY IIa member genes in tandem in antisense and sense orientations with GUS linker in the center. This insert was excited with BamHI and cloned into the Ubi-NC1300 vector (Chern et al. 2005b). The insert sequence is available at GenBank under accession number 1288449.
Rice transformation was as described previously (Chern et al. 2001). Agrobacterium EHA105 was used to infect rice callus. For transformation of Kitaake, hygromycin was used for selection.
RNA extraction and RT-PCR analysis
Total RNA was extracted from leaves using the Trizol reagent (Invitrogen). Total RNA was converted into cDNA using M-MLV reverse transcriptase (EC 18.104.22.168; Invitrogen). PCR analyses were performed with gene-specific primer sets: 5′-ATGGACGACGACGGCGACGGCT-3′/5′- GCGGTCGGCGGCTGCTGTCTC-3′ (for OsWRKY62); 5′- AATGGAGGAAGTATGGTCAGAAGGT-3′/5′- CGTCGATCAGTAGCAATCGTCAAT-3′ (for OsWRKY71); 5′- CATCGTGCCCGGTGAAGAAGAA-3′/5′- AATTCGGGCAGCTTCTGGAGGAT-3′ (for OsWRKY76); 5′- AGAAGGTGACGAAGGACAACCC-3′/5′- GTGACGAGCGCCGCCTTGAAC-3′ (for OsWRKY28); 5′-CGCAGCTCACATTATCAAGTCAGA-3′/5′-GAAGCAGCAATACGGAGATGGATG-3′ (for PR10); 5′- CATGAAGATGCGGACTTACG-3′/5′- ATCCACGCCGTATTCGG-3′ (for GUS). 18S ribosomal RNA was used as an internal control following the QuantumRNA 18S Internal Standards manual (Ambion, Austin, TX, USA). The amplified products were then resolved by gel electrophoresis.
Thanks to Maw-Sheng Chern for valuable discussions and to Brian Conlin for his technical assistance. This work was supported by the National Institute of Health grant #GM59962 to PCR. LEB was supported in part by a University of California Office of the President Postdoctoral Fellowship.
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