A receptor like kinase gene with expressional responsiveness on Xanthomonas oryzae pv. oryzae is essential for Xa21-mediated disease resistance

Leucine-rich repeat receptor-like kinases (LRR-RLKs) represent a large class of proteins in regulating plant development and immunity. The LRR-RLK XA21 confers resistance to the bacterial disease caused by the pathogen of Xanthomonas oryzae pv. oryzae (Xoo). Several XA21 binding proteins have been characterized, however the early events governing XA21 signaling have not been fully elucidated. Here we report the identification of one LRR-RLK gene (XIK1) whose expression is induced rapidly upon the infection with the pathogen of Xoo. Expression pattern analysis reveals that XIK1 is preferentially expressed in reproductive leaves and panicles, and that expression is associated with plant development. By using RNA interference (RNAi), we silenced the expression of XIK1 in rice with Xa21 and found that reduced expression of XIK1 compromised disease resistance mediated by XA21. In addition, we found that the expression of the downstream marker genes of pathogen associated molecular pattern (PAMP) triggered immunity (PTI) in rice was compromised in Xa21 plants silenced for XIK1. Our study reveals that the LRR-RLK gene XIK1 is Xoo-responsive and positively regulates Xa21-mediated disease resistance.


Background
Receptor-Like Kinases (RLKs) represent one of the largest protein families in plants, with more than 600 members in Arabidopsis (Shiu and Bleecker 2001) and 1000 members in rice (Shiu et al. 2004). A typical RLK protein contains extracellular structure, and transmembrane and kinase domains (Greeff et al. 2012). Based on the variation of the N-terminal domains, RLKs are divided into more than 44 subclasses (Gish and Clark 2011). The leucine-rich repeat (LRR)-RLK subclass is the largest, with 165 members in Arabidopsis (Shiu and Bleecker 2001) and 292 members in rice (Shiu et al. 2004). The LRR domain is a 24 residues-containing motif rich for leucine or other hydrophobic amino acids. Each LRR-RLK contains one or more LRRs to form a pocket used for binding to various ligands (Kolade et al. 2006;Gish and Clark 2011).
Xa21 encodes a protein of rice LRR-RLK XII subclass and confers resistance against diverse strains of Xoo (Song et al. 1995). Although the expression of Xa21 is quite stable during rice development, XA21-mediated disease resistance is development-dependent (Mazzola et al. 1994). Previous studies have shown that several XA21 binding proteins (XBs) regulate XA21-mediated immunity. These XBs belong to different protein families: such as RING finger ubiquitin ligase (XB3), transcriptional factor OsWRKY62 (XB10), protein phosphatase 2C (XB15), ATPase (XB24), endoplasmic reticulum chaperone (BiP3) and PANK protein (XB25) (Chen et al. 2010;Peng et al. 2008;Park et al. 2010;Wang et al. 2006;Park et al. 2008;Jiang et al. 2013). Recently, we also reported that OsSERK2, the rice ortholog of BAK1, regulates XA21-mediated immunity in a mechanism distinct from that of BAK1 in regulation of FLS2-and EFR-mediated immunity in Arabidposis (Chen et al. 2014b). Even with these advances, the mechanism of XA21-mediated immunity is still largely unknown.
Here, we characterized XIK1 (LOC_Os02g34790), which encodes a LRR-RLK protein, through analysis of the Rice Oligonucleotide array database (ROAD: http://www. ricearray.org/). We found that the expression of XIK1 was slightly induced upon the inoculation with Xoo in Kitaake but more strongly in Xa21 plants. We found that XIK1 is ubiquitously expressed in different rice tissues and preferentially in leaves and panicles. Transgenic Xa21 plants silenced for XIK1 exhibit compromised disease resistance to Xoo. We also found that the expression of XIK1 is increasing during rice development, which might be the reason why XA21-mediated disease resistance is developmentally dependent. In addition, we found that the expression of marker genes for pathogen associated molecular pattern (PAMP) triggered immunity (PTI) is reduced in Xa21 plants silenced for XIK1. Taken together, our study reveals that the expression of XIK1 is Xoo-responsive and that XIK1 positively regulates XA21-mediated immunity.

Identification of XIK1
LRR-RLK is a large subgroup of the rice RLK family, of which, XA21, XA3/XA26 and OsSERK2 have been reported to regulate plant immunity against Xoo, the causal agent of bacterial leaf blight. To determine if any other LRR-RLK is involved in the immune response against Xoo, we analyzed global transcriptional analysis on the expression pattern of rice LRR-RLK genes to search those with Xoo-responsiveness using the data available from the Rice Oligonucleotide array database (http://www.ricearray.org/). We identified eight LRR-RLK genes whose transcriptional expressions were rapidly (at 2 h post inoculation) up-regulated upon Xoo inoculation (Additional file 1: Figure S1). We then selected one gene, LOC_Os02g34790, for further study. The protein encoded by LOC_Os02g34790 contains a typical signal peptide at the N-terminal, 18 repeated LRR motif and a kinase domain at the C-terminal ( Figure 1). The phylogenetic tree analysis reveals that this protein belongs to LRR II subfamily (Hwang et al. 2011). We then named this gene as Xoo-induced kinase 1 (XIK1).

The expression pattern of XIK1
We performed quantitative RT-PCR to measure the transcription levels of XIK1 in the rice tissues, including root, stem, leaf and panicle at the productive stage. We found that XIK1 was ubiquitously expressed in all of the tissues with a preference in leaves and panicles ( Figure 2A). We then measured the expression of XIK1 in leaves collected from different developmental stages of rice and found that the expression of XIK1 increases during the development Figure 1 Prediction of the amino acid sequence and protein structure of the LRR-RLK XIK1. The sequence from amino acid residue 1 to 26 is predicted as a signal peptide. The extra-cellular region with 17 LRRs consists of the amino acid residues from 28 to 422; the amino acids characterizing the LRR motifs are marked in dark. The transmembrane region consisting of amino acid residues from 486 to 509 are underlined with single solid line. The intracellular kinase domain consisting of the amino acid residues from 550 to 802, with the conserved amino acid residues is indicated by double lines. The structure of XIK1 was predicted according to the structure of XA21 reported previously (G. L. Wang et al. 1996;Xiang et al. 2006). of rice ( Figure 2B). These results suggest that XIK1 may function in both tissue-and development-dependent manners in rice.

The expression of XIK1 in Xa21 plants is induced by Xoo
As the microarray data of XIK1 indicates that the expression of XIK1 in Nipponbare is responsive to Xoo, we tested whether it could also be induced in Xa21 plants . For this purpose, we collected the Xa21 rice leaves post the inoculation with Xoo or H 2 O (mock-treatment) and measured the expression of XIK1 using qRT-PCR. We found that the expression of XIK1 was almost 1.5 folds of that obtained from mock treatment in Xa21 resistant lines ( Figure 3). This result suggests that the expression of XIK1 in Xa21 plants is induced upon the inoculation with Xoo.

Silencing of XIK1 compromises the XA21-mediated resistance to Xoo
Because the LRR-RLK XA21 functions as a pattern recognition receptor (PRR) to confer resistance to Xoo and XIK1 is rapidly induced by Xoo inoculation in Xa21 plants, we presumed that XIK1 regulates XA21-mediated immunity. To test this hypothesis, we introduced the XIK1Ri construct into ProA-Xa21 homozygous rice lines to silence the expression of XIK1 in Xa21 stable transgenic plants. ProA-Xa21 homozygous rice lines confer full resistance to the Xoo strain PXO99A (Chen et al. 2010). We obtained 12 independent double transgenic RNAi lines carrying both Xa21 and XIK1Ri and named them Xa21XIK1Ri (XXIK1Ri) lines. Three representative lines with reduced expression levels of XIK1 and stable expression level of the XIK1 homolog LOC_Os02g34750 were found (Additional file 2: Figure S2). Compared with that in the wild type ProA-Xa21 plants, the expression of XIK1 is reduced and is about 40%, 45% and 80% in the three XXIK1RNAi lines, XXIK1Ri-1, XXIK1Ri-3 and XXIK1Ri-8, respectively. Expression of LOC_Os02g34750, with the highest identity (80.1%) of cDNA sequence to LOC_Os02g34790, was not altered (Additional file 2: Figure S2). We inoculated the T0 generations of these transgenic lines, and observed that the Xa21 plants  silenced for XIK1 exhibited compromised disease resistance to the Xoo strain PXO99A compared with the wild type ProA-Xa21 plants (Additional file 3: Figure S3A-C). We then analyzed the disease resistance of plants from T1 generations after inoculation with PXO99A. We found that the T1 plants segregated with partial resistance in some segregants and typical full resistance in others. The double transgenic plants carrying Xa21 and silenced for XIK1 are partially resistant, showing typical long water-soaked lesions. The segregants carrying Xa21 but lacking of XIK1Ri confer full resistance to Xoo as do the wild type Xa21 plants (Additional file 3: Figure S3D). The Xa21 plants are highly resistant and the Kitaake plants are highly susceptible to the Xoo strain PXO99A as expected.
To characterize the effect of XIK1 on XA21-mediated disease resistance, we performed more detailed investigations using the plants (XXIK1Ri) homozygous for both Xa21 and XXIK1Ri derived respectively from three of the double transgenic lines. After we verified that the expression of XIK1 was reduced whereas the expression of Xa21 was not obviously changed in these RNAi lines (Additional file 4: Figure S4), we inoculated the plants with the Xoo strain PXO99AZ. At 14 days post inoculation (DPI), all of these three XXIK1Ri lines displayed longer lesions than the Xa21 control ( Figure 4A-E). The average lesion length of the line XXIK1Ri-1 with the lowest expression level of XIK1 is about 4.29 ± 0.69 cm, with the longest lesion length among these three represent XIK1 silence lines. Meanwhile, XXIK1Ri-8 with the highest expression of XIK1 showed the shortest lesion length (3.29 ± 0.72) cm among these three XIK1Ri lines. The positive control Xa21 plants and the negative control Kitaake plants respectively show the average lesion length of 0.26 ± 0.10 cm for the full resistance and 7.33 ± 0.10 cm for the full susceptibility as expected ( Figure 4F). These results suggest that these RNAi transgenic plants exhibit partial resistance, showing typical longer water-soaked lesions than ProA-Xa21 plants but shorter than Kitaake plants.
We then quantified bacteria accumulation in plant leaves after inoculation with Xoo at 0, 7, 14 and 21 DPI. We found that, at 21 DPI, the bacterial numbers in XXIK1Ri lines were almost 100 fold (4.8×10 11 for XXIK1Ri-1, 2.0×10 11 for XXIK1Ri-3 and 1.4×10 11 for XXIK1Ri-8) greater than in ProA-Xa21 control plants (2.8×10 8 ) ( Figure 4G). These results are consistent with the results of disease lesion length data described above. The lesion lengths are longer and the bacterial numbers are greater when the expression level of XIK1 is lower in the XXIK1Ri lines (Additional file 2: Figure S2 and Additional file 4: Figure S4), suggesting that the resistance of the transgenic lines is in accordance with the expression level of XIK1. Taken together, these results suggest that reduced expression of XIK1 compromises XA21-mediated disease resistance and the LRR-LRK XIK1 plays essential roles in XA21-mediated full innate immunity in rice.
Reduced expression of XIK1 compromises the downstream response of PTI Previous reports have shown that the genes, OsKS4 (LOC_Os04g10060) and Os04g10010(LOC_Os04g10010), function as marker genes involved for the downstream responses associated PTI (Park et al. 2012;Chen et al. 2014a). Thus, we asked whether these two genes in Xa21 plants are also responsive to Xoo. For this purpose, we measured the expression of these two genes in rice plants post the inoculation with Xoo or H 2 O as a mock treatment. Compared with mock treatment, the expression levels of OsKS4 and Os04g10010 were induced more than two folds at 2 days post Xoo treatment ( Figure 5). This result suggests that the XA21-mediated immune signaling was transduced downstream quickly. We then performed qRT-PCR analysis on the expression of the two genes using the leaf tissue samples collected from Kitaake, ProA-Xa21, and XXIK1Ri-1 plants without Xoo inoculation. We found that the expression levels of these two genes were respectively higher in ProA-Xa21 plants than that in Kitaake plants, suggesting that rice plants carrying Xa21 possess a higher level of basal PTI than Kitaake plants. More interestingly, we found that the expression of the two genes, OSKS4 and Os04g10010, were much reduced in XXIK1Ri-1 plants (3.6 ± 0.4 fold and 2.8 ± 0.2 fold, respectivly) compared to ProA-Xa21 plants (8.5 ± 0.1 fold and 4.0 ± 0.7 fold, respectivly) ( Figure 6A). We then measured the expression of OSKS4 and Os04g10010 in ProA-Xa21 and XXIK1Ri-1 plants after Xoo inoculation. We found that the induction of both genes were inhibited in the plants silenced for XIK1 ( Figure 6B and C). These results suggest that reduced expression of XIK1 compromises XA21-mediated immune response both before and after Xoo inoculation.

Expression of the LRR-RLK gene XIK1 is responsive to Xoo inoculation
Although some resistant proteins have been reported to regulate rice resistance against Xoo, prior studies of RLK family proteins in rice were mainly focused on two RLKs: XA21 and XA3/XA26 (Chen and Ronald 2011). Phosphorylation events play critical roles for these RLKs to initiate the response triggered by PAMPs (Chen et al. 2010;Chen et al. 2014a). There are few reports showing that the expression of these RLKs is induced by pathogens and thus regulates the immune response (Century et al. 1999). Our study reveals that the RLK gene XIK1 was induced by Xoo and that the encoded protein XIK1 positively regulates XA21-mediated immunity (Figures 3 and 4). Previous studies have identified some proteins that play important roles in the regulation of XA21-mediated resistance. For examples, ubiquitin E3 ligase XB3 ) and endoplasmic reticulum chaperone BiP3 were shown to keep the XA21 protein stable (Park et al. 2014). Protein phosphatase 2C XB15 (Park et al. 2008), ATPase XB24 and rice somatic embryogenesis receptor-like kinase 2 (OsSERK2) regulate the phosphorylation of XA21 (Chen et al. 2010;Chen et al. 2014a), and the WRKY transcriptional factor XB10 regulates the XA21-mediated immune response through an unknown mechanism . However, the expressions of these genes were not obviously changed upon the inoculation of the Xoo strain PXO99A. Our finding that the expression of XIK1 was induced by Xoo suggests that some RLKs such as XIK1 regulate PTI via their transcriptional expression (Figure 3). This finding provides new insights in understanding the XA21-mediated PTI signaling and the RLK-mediated innate immunities. We also found that the PTI-related marker genes were down regulated in Xa21 plants silenced for XIK1 (Figures 5 and 6), suggesting that the expression of the genes involved in downstream of XA21-signaling are regulated by XIK1. Additionally, since expression of XIK1 is both tissue-and developmentally-dependent indicates that XIK1 regulates rice development and immunity tissue-specifically and developmentally ( Figure 2). This result also provides the explanations why the disease resistance mediated by XA21 is developmentally-dependent.
We also performed a similar Xoo inoculation assay on four RNAi lines silenced for XIK1 in Kitaake rice and found that these RNAi lines were as susceptible as Kitaake plants (data not shown). Thus, our study suggests that XIK1 regulation of rice resistance to Xoo is XA21-dependent.

XIK1 regulates early events in XA21-mediated immunity signaling
LRR-RLKs consist of the largest RLK subfamily in plants with more than 165 members in Arabidopsis (Shiu and Bleecker 2001) and more than 292 members in rice (Shiu et al. 2004). In rice, the well studied LRR-RLK is XA21. Investigation of the molecular mechanisms of XA21-mediated immunity have shown that many proteins are required for XA21 to function Park et al. 2010;Wang et al. 2006;Park et al. 2008;Jiang et al. 2013;Chen et al. 2014a). Our study reveals that the LRR-RLK XIK1 positively regulates XA21-mediated immune signaling. As the expression of XIK1 is induced as early as within 2 h post Xoo inoculation and reduced expression of XIK1 compromises the expression of PTI-related genes, we hypothesize that XIK1 regulates the early events of XA21-mediated signaling. Because the XIK1 shares structural similarity with the LRR-RLK XA21, such as the extracellular LRR domain, a transmembrane domain that is required for the plasmembrane-localization of RLK proteins, and the intercellular active kinase domain, we suggest that XIK1 might work as a co-receptor of XA21 for PAMPs recognition and mediate the downstream signaling. As we could not detect the interaction between XIK1 and XA21 by using the intercellular domains of these two Figure 5 Expression of two PTI marker genes is induced within 2 h after Xoo inoculation. OSKS4 and Os04g10010 are rapidly induced after the infection of Xoo PX099A was used. Expression levels for each gene were normalized to the expression of the ubiquitin5 reference gene. Bars depict average expression level ± SD of three technical replicates. The ** indicate significant differences (P<0.05) as determined by a one-way ANOVA followed by post hoc Tukey HSD analysis. This experiment was repeated three times with similar results.
(See figure on previous page.) Figure 4 Determination of disease resistance of ProA-Xa21 plants silenced for XIK1. Six week-old plants were inoculated with the Xoo strain PXO99A. The ProA-Xa21 and Kitaake (Kit) were used as resistant and susceptible controls, respectively. Photograph depicts the representative leaves from plants at 14 DPI. The leaves from Kitaake displayed the full susceptibility to Xoo (A) whereas the leaves from ProA-Xa21 displayed full resistance to Xoo (B). The panels C, D, and E show the representative leaves from the three transgenic lines, XXIK1Ri-1, XXIK1Ri-3 and XXIK1Ri-8, respectively. All photographs were taken at 14 DPI. Bars= 5cm. (F), Lesion lengths of XXIK1Ri after inoculation with Xoo. Lesion length was measured at 14 DPI. Graph shows average lesion length ± SD of at least 20 leaves from 10 independent plants homozygous for both Xa21 and XIK1Ri. The letters indicate significant differences (P<0.05) as determined by a one-way ANOVA followed by post hoc Tukey HSD analysis. (G), Bacterial cell numbers accumulated in ProA-Xa21 plants silenced for XIK1 after inoculation with Xoo. Bacterial cell numbers were counted at day 0, 7, 14, 21 post inoculations. Each data point represents the average ± SD of six leaves from two independent plants. The ** indicate significant differences (P<0.05) as determined by a one-way ANOVA followed by post hoc Tukey HSD analysis. These experiments were repeated at least three times with similar results.
RLKs in yeast (data not shown), the extracellular domains of XIK1 and XA21 or the binding of the PAMP might be required for their interaction. To understand the precise mechanism of XIK1 in regulation of XA21-mediated immunity, it is interesting to address these questions in our future work, including whether XIK1 forms a complex with XA21, whether the extra-cellular domains of these two RLKs, or pathogen inoculation are required for their interaction, and whether OsSERK2, a recently identified RLK essential for XA21-mediated immunity is required for XIK1 to function. We also suggest that XIK1 may regulate XA3/XA26, mediated immune response to Xoo, as XA3/XA26 belongs to LRR XII RLK with the similar structure of XA21.
In Arabidopsis, the LRR-LRKs, FLS2 and EFR also mediate PTI by recognizing the PAMPs, flagellin and elongation factor (EF-tu), respectively (Gomez-Gomez and Boller 2000;Zipfel et al. 2006). Previous studies have shown that FLS2-and EFR-mediated immunities share many similar processes as XA21, such as production of reactive oxygen species (ROS), activation of mitogen activated protein kinases (MAPKs), induction of pathogenesis-related genes (PRs) (Gomez-Gomez et al. 1999;Zhang and Zhou 2010;Zipfel et al. 2006) and the requirement of the SERK proteins (i.e. SERK3/BAK1 and SERK4/BKKI for FLS2 and EFR in Arabidopsis, and OsSERK2 for XA21 in rice) for their early signaling (Korner et al. 2013;Yang et al. 2011;Chen et al. 2014a). We hypothesize that the RLKs, encoded by At01g35710 and At04g08850, with high identities of amino acid sequence to XIK1, might also regulate the FLS2-and EFR-mediated early signaling through a similar mechanism as that of XIK1.
LysM-RLKs, another RLK subgroup, have been shown to mediate PTI in rice and Arabidopsis (Miya et al. 2007;Shimizu et al. 2010;Chen and Ronald 2011). The LysM conserved motif of these receptors is reported to mediate plant-pathogen interactions by recognizing chitin molecules derived from fungal pathogens such as Magnaporthe oryzae (Chen and Ronald 2011). Thus, it would also be of great interest to determine whether XIK1 or other proteins with a similar structure of XIK1 regulate LysM-RLK-mediated immunity in the future work.

Conclusion
Our study revealed that the LRR RLK gene XIK1 is pathogen responsive and its expression is induced rapidly upon the inoculation with Xoo. Silencing of XIK1 comprises XA21-mediated resistance to Xoo and XIK1 positively regulates XA21-mediated resistance.

Plant growth, Xoo inoculation and disease resistance determination
All of the plants were grown in rice fields (Wenjiang, Chengdu, Sichuan, China). Rice Kitaake is used for transcriptional expression pattern analysis. The silencing construct of XIK1 (XIK1RNAi) was introduced into the Xa21 (ProA-Xa21) plants with full resistance to the Xoo strain PXO99A (Hopkins et al. 1992) to obtaine double transgenic plants containing Xa21 and XIK1RNAi. Plants were inoculated with the Xoo strain PXO99A. The inoculation and disease lesion length determination was performed according to the methods described previously (Song et al. 1995). Statistical analysis was performed using a one-way ANOVA followed by post hoc Tukey HSD. The leaves inoculated with Xoo were collected and prepared for transcriptional expression analyses as described below. Figure 6 Silencing of XIK1 reduces the basal level of PTI marker genes in ProA-Xa21 plants. The relative expression level of two rice PTI marker genes, OSKS4 and Os04g10010, were determined by qRT-PCR using RNA extracted from rice leaves of six weeks old plants without Xoo inoculation (A) and 2 days post Xoo inoculation (B, C). All data were normalized to the expression of the ubiqutin5 reference gene. The results of one representative experiment were shown. Error bars indicating SD of three technical replicates. Three independent biological experiments were repeated with the similar results. The letters indicate significant differences (P<0.05) for each gene determined by a one-way ANOVA followed by post hoc Tukey HSD analysis.

RNA extraction and real time RT-PCR analyses
Total RNA was isolated from rice plant tissues using Invitrogen RNA isolation reaction, TRIzol (Invitrogen), following the procedures of the manual. The first strand cDNA was synthesized using the Takara reverse transcription kit (Takara). Quantitative real time PCR (qRT-PCR) was performed on a Bio-Rad CFX96 Real-Time System coupled to a C1000 Thermal Cycler (Bio-Rad). For qRT-PCR reactions, the SsoFastEva Green Super mix was used. The gene expression levels relatively to the ubiquitin5 gene (LOC-06g46770) were analyzed using delta-delta ct method. All of the qRT-PCR primers are shown in Additional file 5: Table S1.