Plant Physiology and Biochemistry 73 (2013) 383e391

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Research article

Arabidopsis cysteine-rich receptor-like kinase 45 positively regulates disease resistance to Pseudomonas syringae Xiujuan Zhang a, b,1, Xiaomin Han a,1, Rui Shi a, Guanyu Yang a, Liwang Qi c, Ruigang Wang a, Guojing Li a, * a b c

College of Life Sciences, Inner Mongolia Agricultural University, Hohhot, Inner Mongolia 010018, PR China Inner Mongolia Institute of Biotechnology, Hohhot, Inner Mongolia 010070, PR China The Research Institute of Forestry, The Chinese Academy of Forestry, Beijing 100091, PR China

a r t i c l e i n f o

a b s t r a c t

Article history: Received 12 April 2013 Accepted 18 October 2013 Available online 26 October 2013

Arabidopsis cysteine-rich receptor-like protein kinase 45 (CRK45) was found to be involved in ABA signaling in Arabidopsis thaliana previously. Here, we reported that it also positively regulates disease resistance. The CRK45 overexpression plants increased expression of the defense genes, and enhanced resistance to Pseudomonas syringae whereas the crk45 mutant were more sensitive to P. syringae and weakened expression of the defense genes, compared to the wild type. We also found that treatment with P. syringae leads to a declined expression of CRK45 in the npr1 mutant and the NahG transgenic plants. At the same time, significantly decreased expression of CRK45 transcript in the wrky70 mutant than that in the wild type was also detected. Our results suggested that CRK45 acted as a positive regulator in Arabidopsis disease resistance, and was regulated downstream of NPR1 and WRKY70 at the transcriptional level. Ó 2013 Elsevier Masson SAS. All rights reserved.

Keywords: Arabidopsis thaliana CRK45 NPR1 Pseudomonas syringae Receptor-like protein kinase Salicylic acid WRKY70

1. Introduction Plants often suffer from various attacks from pathogens and they have evolved an array of complicated defense mechanisms that are activated by multiple defense signaling pathways. During the defense responses, plants have a common feature: activating a large number of genes after pathogen infection or treatment with pathogen elicitors [1]. In this process, salicylic acid (SA) plays a prominent role as a signal molecule. When treated with exogenous SA or encountering pathogen infection, SA accumulated in local and

Abbreviations: AIG1, AvrRpt2-induced gene 1; CRKs, cysteine-rich receptor-like kinases; DIG, digoxigenin; JA, jasmonic acid; LAR, local acquired resistance; MeJA, methyl jasmonic acid; NPR1, non-expressor of PR genes 1; PR1, pathogenesisrelated gene 1; PR2, pathogenesis-related gene 2; Pst DC3000, Pseudomonas syringae pv. tomato DC3000; RLKs, receptor-like kinases; SA, salicylic acid; SAR, systemic acquired resistance; TF, transcription factor; TGA, TGACG sequencespecific binding protein; WRKYs, WRKY DNA binding proteins. * Corresponding author. Tel.: þ86 471 4304172. E-mail addresses: [email protected] (X. Zhang), hanxiaominhushi@163. com (X. Han), [email protected] (R. Shi), [email protected] (G. Yang), [email protected] (L. Qi), [email protected] (R. Wang), liguojing@imau. edu.cn (G. Li). 1 Authors contributed equally to this work. 0981-9428/$ e see front matter Ó 2013 Elsevier Masson SAS. All rights reserved. http://dx.doi.org/10.1016/j.plaphy.2013.10.024

systemic tissues, and the systematic acquired resistance (SAR) usually established, leading to the expression of a set of PATHOGENESIS-RELATED (PR) genes and production of defense proteins [2]. NONEXPRESSOR OF PR GENES 1 (NPR1), a cytosolic protein, is considered to be the crucial component of the SA signaling pathway [3]. In response to increased SA levels, NPR1 interacting with TGA transcription factors are necessary for PR1 gene expression [3,4]. At the same time, several members of the WRKY family also act as mediators of pathogen-associated transcriptional reprogramming in plants [5]. The WRKY transcription factor family is specific to plants and appears to be involved in the regulation of plant defense reaction [6,7]. The Arabidopsis genome encodes totally 74 WRKYs, and members of this family contain at least one conserved DNA-binding region, designated as the WRKY domain, which contains a conserved WRKYGQK sequence followed by a Cys2His2 or Cys2HisCys zinc-binding motif, and specifically recognize the Wbox sequences (TTGAC) located in the promoter region of most defense-related genes [6,8,9]. Many WRKYs encoding genes are rapidly induced after treatment with elicitors or after infection by pathogens [10]. One member of the WRKY family, WRKY70, has been functionally characterized as a positive regulator in the SAmediated gene expression and disease resistance of plants and as a negative regulator in SA biosynthesis [11].

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Fig. 1. The expression pattern of CRK45 after treated by P. syringae, SA, MeJA and B. cinerea. (A) RNA gel blot analysis showing the expression of CRK45 in wild type and the crk45 mutant after Pst DC3000 and Pst AvrRpm1 treatment. (B) RNA gel blot analysis showing the expression of CRK45 in wild type Arabidopsis after SA or MeJA treatment. After pathogen inoculation or spraying with 1 mM SA or MeJA, total RNA was extracted from leaves of four-week-old plants grown in pots containing soil mixture and samples were collected at the indicated times. Ten micrograms of RNA was loaded per lane, and the CRK45 gene-specific probe was used for hybridization. (C) The expression of CRK45 in wild type after B. cinerea treatment. Leaves of two-week-old seedlings growing in GC vials were inoculated with 4  105 spores per vial. Samples were harvested 3 or 8 h after inoculation and total RNA was extracted for qRT-PCR analysis. EF1a was chosen as an internal control. Expression level for each gene was normalized to that of the control. For all experiments, three independent experiments were performed, and each data point represents the average of three technical replicates  SD.

Receptor-like kinases (RLKs) belong to one of the largest gene families in plants. For instance, there are more than 610 members in Arabidopsis. RLKs are well known as conserved signaling components that regulate development, disease resistance, hormones perception, and self-incompatibility in plants [12,13]. Cysteine-rich receptor-like kinases (CRKs) are one of the largest RLK groups with 44 members, which have been suggested to play important roles in regulation of defense responses and programmed cell death [14]. Most of the CRKs are induced by pathogen attack and application of SA at the transcriptional level [15,16]. Accordingly, several CRKs are involved in regulation of defense and cell death in Arabidopsis. For example, constitutive over-expression of CRK5 (At4g23130) led to increased resistance to the virulent bacterial pathogen Pseudomonas syringae pv. tomato DC3000 (Pst DC3000) [16]. Overexpression of CRK13 (At4g23210) resulted in enhanced resistance to P. syringae and increased SA content [17], while CRK11 was reported to take part in the interaction between pathogen and Arabidopsis and in defense responses [15]. In this study, we found that one member of the CRKs family, At4g11890, named as CRK45 [14] or ARCK1 [18], is involved in disease resistance. Overexpression of CRK45 resulted in resistance to the virulent bacterial pathogen strain Pst DC3000 and elevated expression of the pathogenesis-related (PR) genes, while the crk45 mutant conferred the contrary phenotypes with slightly declined expression of the PR genes, indicating that CRK45 positively regulates Arabidopsis disease resistance. 2. Results 2.1. CRK45 is induced by P. syringae and salicylic acid In a microarray analysis aimed to identify Arabidopsis genes that are induced in response to infection by P. syringae [19,20], CRK45 was identified as one of the early pathogen-responsive genes. To confirm that CRK45 was indeed one of the pathogen-responsive genes, we detected the CRK45 transcript after P. syringae pv. tomato DC3000 (Pst DC3000) and Pst AvrRpm1 inoculation. The

results showed that CRK45 were induced by both Pseudomonas strains (Fig. 1A). At the same time we also detected the CRK45 transcript after Botrytis cinerea (B.c.), a necrotroph fungi, infection, and similar results were obtained (Fig. 1C). This suggested that CRK45 might have functions during interaction of plants with a broad-spectrum of pathogens. In addition, CRK45 was also responsive to SA treatment (Fig. 1B). While after MeJA treatment, the transcript induction of CRK45 has no obvious changes compared with the PDF1.2 gene expression as a control (Fig. 1B and S1), indicating that this gene responded only to pathogens. In our previous studies, we had obtained a CRK45 T-DNA insertion line (Salk-057538) from the Arabidopsis Biological Research Center, and the CRK45 overexpression plants were also generated by putting the CRK45 genomic DNA sequence under the control of the 35S promoter from cauliflower mosaic virus [21]. To further determine the biological function of CRK45 in plant defense response, we generated the CRK45 complementary plants as well by putting the CRK45 genomic DNA sequence under the control of the 35S promoter and transformed it into the crk45 mutant. As shown in Fig. 1A, the transcript of CRK45 was undetectable by Northern blotting in the crk45 mutant, indicating that the T-DNA insertion resulted in completely knock-out of CRK45. The expression level of CRK45 showed different level of increase in both the overexpression and complementary lines (OE-15, OE-37 and com-17), and these transgenic lines were used in the following experiments unless stated otherwise (Fig. 2S). The expression of CRK45 in different tissues including siliques, leaves, stem and flowers were detected, and the results showed that the CRK45 transcript expressed at a higher level in leaves than in other tissues (Fig. 3S). 2.2. Knock out of CRK45 resulted in sensitivity to P. syringae To determine the contribution of CRK45 to disease resistance in Arabidopsis, we sprayed Pst DC3000 (108 cfu mL1 (OD600 ¼ 0.1)) to Col-0 (wild type), the crk45 mutant, CRK45 overexpressing plants,

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Fig. 2. The phenotypes of wild type, the crk45 mutant, CRK45 overexpression lines and NahG transgenic plants after spraying with Pst DC3000. (A) and (B), Disease symptoms of plants from four different genotypes caused by Pst DC3000 infection. Leaves of 4-week-old plants of wild type, the crk45 mutant, CRK45 overexpression lines and NahG transgenic plants were sprayed with 108 cfu mL1 of Pst DC3000 and photographs were taken 3 days after the spray. The whole plants were shown in (A), and a close-up of the diseased leaves was shown in (B). (C) The bacterial growth assay among the four genotypes after Pst DC3000 inoculation. Leaves from 4-week-old plants of wild type, the crk45 mutant, CRK45 overexpression lines, CRK45 complementary line, and NahG transgenic plant were inoculated with 106 cfu mL1 of Pst DC3000, colony-forming units (cfu) were calculated at 0, 2 and 4 days after inoculation. The experiments were repeated twice, and similar results were obtained. Letters indicate differences at P < 0.05 (n ¼ 3).

and NahG transgenic plants (which carried the salicylate hydroxylase encoding NahG transgene, and could convert SA into catechol, hence is defective in non-host resistance to pathogen [22]) was used as control. Three days after the spray, the crk45 mutant and

NahG transgenic plants appeared obvious disease symptoms compared with wild type (Fig. 2, A and B). On the contrary, the disease symptom of the CRK45 overexpressing plants was less severe than that of wild type.

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Fig. 3. The expression pattern of pathogenesis-related genes in wild type, the crk45 mutant, CRK45 overexpression lines and CRK45 complementary lines after Pst DC3000 and SA treatment. (A), (B) and (C), Expression of PR1, PR2 and AIG1 were analyzed by qRT-PCR after Pst DC3000 treatment. Leaves of 4-week-old wild type, the crk45 mutant, CRK45 overexpression lines and CRK45 complementary line were inoculated with 5  107 cfu mL1 of Pst DC3000. Samples were harvested after eight hours and total RNA was extracted for qRT-PCR analysis. EF1a was selected as an internal control. Expression level for each gene was normalized to that of the wild type. For all experiments, three independent experiments were performed. Each data point represents the average of three technical replicates  SD. (D) Expression of PR1 was analyzed by Northern blotting after SA treatment. Leaves of 4-week-old wild type, the crk45 mutant and CRK45 overexpression lines were sprayed with 1 mM of SA. Samples were harvested at indicated times and total RNA was extracted for Northern blotting analysis. Gene-specific probes were labeled with DIG (digoxigenin). Two independent experiments were performed with similar results obtained.

In planta bacterial growth assay was then conducted among the four genotypes after Pst DC3000 inoculation, and the results were consistent with the disease symptoms: the CRK45 overexpression lines supported lower number of bacterial growth compared with wild type, and the crk45 mutant showed the opposite responses (Fig. 2C). These results suggested that CRK45 positively regulated Arabidopsis resistance to P. syringae. 2.3. Altered expression of CRK45 affects the expression of pathogenesis-related genes after Pseudomonas or SA treatment Enhanced disease resistance in Arabidopsis is often accompanied by the accumulation of PR genes’ transcripts. The expression level of PR genes, including PR1, PR2 and AIG1, were analyzed using real-time RT-PCR. As shown in Fig. 3, the expression of all PR genes in the CRK45 overexpression lines was higher and peaked earlier than that in wild type after Pst DC3000 inoculation, whereas in the crk45 mutant, the transcript induction of PRs was slightly lower after inoculation. The expression pattern in CRK45 complementary plant was similar to the overexpression lines or wild type (Fig. 3, com17). Since CRK45 was also induced after SA treatment, the expression of PR1 after SA treatment in different genotypes was detected accordingly. The results were similar to that treated by Pst DC3000, and the transcript level of PR1 significantly higher in CRK45 overexpression lines than that in wild type (Fig. 3D). Therefore, the results suggested that CRK45 played a positive role in disease resistance by affecting the expression of the PR genes.

2.4. Knock-out of CRK45 slightly decreased salicylic acid biosynthesis Previous results have shown that the function of CRK45 in disease resistance maybe relate to the SA-dependent resistance signaling pathways. ICS1, which encoding the isochorismate synthase, was required to synthesize SA and for local acquired resistance (LAR) and SAR responses [23]. Meanwhile, EDS5, as a putative SA transporter, have an essential function in the SA-dependent pathway for plant defense against pathogens [24]. The expression levels of ICS1 and EDS5 in wild type, the crk45 mutant, the CRK45 overexpression lines and the CRK45 complementary line were detected after Pst DC3000 treatment. The results showed that these two genes were significantly induced in the CRK45 overexpression lines compared with that in wild type, conversely in the crk45 mutant their expression was lowered (Fig. 4, A and B), suggesting that CRK45 is involved in the SA-dependent disease resistance signaling pathways. We then determined the SA contents after inoculation with Pst DC3000. SA accumulated at a lower level in the crk45 mutant compared to the wild type plants (Fig. 4C). This suggests that CRK45 regulates defense response by affecting SA accumulation. 2.5. NPR1 and WRKY70 positively regulated CRK45 at the transcriptional level Previous studies showed that in SA-dependent resistance signaling pathways, NPR1 functions as an important signal

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molecule. The npr1 mutant was a typical Pst DC3000 susceptible mutant and was confirmed to have little expression of PRs [25]. From the Gene Expression Omnibus (GEO) database we found that the expression level of CRK45 was reduced in the npr1 mutant (http://affymetrix.arabidopsis.info/narrays/experimentpage.pl? experimentid ¼ 355). Our results further confirmed that the CRK45 transcript was obviously diminished in the npr1 mutant after Pst DC3000 treatment (Fig. 5). In addition, we also found that the expression of CRK45 declined remarkably after Pst DC3000, Pst AvrRpm1 and SA treatment in the susceptible NahG transgenic plant, similar to that in the npr1 mutant (Fig. 5). These results suggested that CRK45 played its role downstream of NPR1. We also found the expression of NPR1 transcript was obviously lowered in NahG transgenic plant than in Col-0 after Pst DC3000 treatment, but its level in the crk45 mutant is only slightly declined compared to that in Col-0 (Fig. 4S). This result explained the more sensitive disease phenotype in NahG transgenic plant than that in the crk45 mutant (Fig. 2). Except for the PR genes, NPR1 also adjusted a handful of downstream genes expression, including some of the TGA (TGACG sequence-specific binding protein) family genes and WRKY (WRKY DNA binding protein) family genes [10,26]. In order to further determine the function of CRK45 in disease signaling pathway, we tested the expression of TGA family genes (including TGA2, 3, 5 and 6) and WRKY family genes (including WRKY18, 25, 38, 54, 62 and 70) in wild type, the crk45 mutant and the CRK45 overexpression lines after Pst DC3000 treatment. The results showed that the expression of TGAs have no significant difference among the three genotypes (Fig. 5S). But in WRKY family, the expression of most of the selected genes, such as WRKY18, 38, 62 and 70, was significantly higher in CRK45 overexpression lines, but only dropped slightly in the crk45 mutant (Fig. 6). According to the previous researches, the transcript of WRKY70 was significantly declined in the npr1 mutant and the NahG transgenic plant than that in wild type [27]. In our experiments, the expression of WRKY70 was significantly reduced in the npr1 mutant

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Fig. 5. The expression of CRK45 in disease sensitive mutant npr1 after Pst DC3000, Pst AvrRpm1 or SA treatment. Leaves of 4-week-old wild type, the npr1 mutant and the NahG transgenic plants were inoculated with 5  107 cfu mL1 of Pst DC3000, Pst AvrRpm1 or sprayed with 1 mM SA. Samples were harvested at indicated times and total RNA was extracted for Northern blot analysis. Ten micrograms of RNA was loaded per lane, and the CRK45 gene-specific probe labeled with DIG was used for hybridization. Two independent experiments were performed and the results were similar.

after Pst DC3000 treatment, confirmed the previous reports (Fig. 7A). Interestingly, the expression of CRK45 in the wrky70 mutant was also suppressed after Pst DC3000 treatment, similar to the expression pattern in the npr1 mutant (Fig. 7B), suggesting that WRKY70 also positively regulated the transcriptional expression of CRK45. 2.6. CPR5 negatively regulated CRK45 at transcriptional level The cpr5 mutant, which was a typical constitutive disease resistance mutant, was found to have constitutive PR1 expression and a higher level of SA [28]. The GEO database also indicated that the expression level of CRK45 was increased in the cpr5 mutant. Our experiments proved that the transcript of CRK45 was truly elevated after Pst DC3000 treatment in the cpr5 mutant (Fig. 7B). In addition, the transcript of WRKY70 was also obviously higher in the cpr5 mutant than that in wild type (Fig. 7A). The expression pattern of

Fig. 4. The induction of SA biosynthetic genes and SA accumulation after Pst DC3000 inoculation. (A) The expression of ICS1 and EDS5 in wild type, the crk45 mutant, CRK45 overexpression lines and CRK45 complementary lines after Pst DC3000 treatment. Leaves of 4-week-old plants were inoculated with 5  107 cfu mL1 of Pst DC3000. Samples were harvested three hours after inoculation and total RNA was extracted for qRT-PCR analysis. EF1a was chosen as an internal control. Expression level for each gene was normalized to that of wild type. For all experiments, three independent experiments were performed, and each data point represents the average of three technical replicates  SD. (B) The content of free SA and total SA in the un-inoculated and Pst DC3000-inoculated leaves of wild type and the crk45 mutant was quantified by HPLC (see Materials and methods). FW means fresh weight. Two independent experiments were performed and the results were similar.

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Fig. 6. Altered expression of CRK45 affects the expression of WRKYs after Pst DC3000 treatment. Leaves of 4-week-old wild type, the crk45 mutant, and CRK45 overexpression lines were inoculated with 5  107 cfu mL1 of Pst DC3000. Samples were harvested after three hours and total RNA was extracted for qRT-PCR analysis. EF1a was used as an internal control. Expression level for each gene was normalized to that of the wild type. For all experiments, two independent experiments were performed. Each data point represents the average of three technical replicates  SD.

WRKY70 in different mutants was similar to that of CRK45 (Fig. 7A and B), further supporting that CRK45 works downstream of WRKY70 in the same signaling pathway. 3. Discussion 3.1. The expression of CRK45 CRK45 is a member of cysteine-rich receptor-like kinases (CRKs) family, which is one of the largest RLK groups with 44 members. According to phylogenetic tree constructed by Wrzaczek [14], CRK45 has the furthest relationship with other members in the family. The double mutant of CRK45 and CRK36 did not show more sensitive phenotype than single mutants after ABA treatment [18]. On these grounds the phenotype of the CRK45 overexpression lines were caused by overexpression of CRK45 rather than gene redundancy. 3.2. CRK45 positively regulates defense response in Arabidopsis As one member of the receptor-like protein kinase family, CRK45 was reported to function in response to ABA and abiotic stresses in Arabidopsis [18,21]. In this study, we found that overexpression of CRK45 enhanced Arabidopsis resistance to P. syringae, while the crk45 mutant appeared sensitive phenotype (Fig. 2), indicating that CRK45 acts as a positive regulator in plant immunity. In expression assays of pathogenesis-related genes, PRs were significantly increased in the CRK45 overexpression lines and reduced slightly in the crk45 mutant after P. syringae or SA treatment (Fig. 3). These data suggested that the constitutive activation of CRK45 controlled defense mechanisms directly contributes to the enhanced disease resistance. 3.3. A working model of CRK45 in plant defense response The results presented here argue for a model we proposed (Fig. 8): In which recognition of a pathogen elevated the expression of ICS1 and EDS5 [23,24,28], and resulted in SA biosynthesis and the

expression of PRs genes [23,24]. Accumulation of SA activated NPR1 and its downstream genes expression [25]. In our studies, the CRK45 transcript was repressed in the npr1 mutant and the NahG transgenic plant (Fig. 5). Thus we presumed that CRK45 should function downstream of NPR1. Our further experiments proved that the expression of CRK45 also declined in the wrky70 mutant after Pst DC3000 treatment (Fig. 7B), suggesting that WRKY70 could regulate the expression of CRK45 as well. Previous studies and our results all pointed out that NPR1 directly regulated WRKY70 expression [27], and in the npr1 mutant, the transcript of WRKY70 was almost undetectable (Fig. 7A). So we get conclusion that NPR1 regulates WRKY70, while WRKY70 regulates CRK45 transcription directly downstream. This model explains why the expression patterns of both WRKY70 and CRK45 was strikingly similar in the npr1 mutant (Fig. 7). Analysis of the CRK45 promoter shows that it contains one W-box located 138 bp upstream of the start codon ATG. Therefore, WRKY70 maybe act on the W-box of the CRK45 promoter so as to regulate its expression. For the negative regulator CPR5, our evidence showed that it inhibited the expression of both WRKY70 and CRK45 (Fig. 7). SNI1 (suppressor of nonexpressor of PR genes 1, inducible 1), another negative regulator of the PR1 expression, its mutant sni1 showed resistance to P. syringae [29,30]. In the sni1 mutant, CRK45 has higher expression than in wild type [30], suggesting that SNI1 also blocked the expression of CRK45 by acting upstream of NPR1 (Fig. 8). Although the transcriptional regulation of CRK45 seems to appear a clear clue, we could not ignore that the function of CRK45 was far beyond fully revealed. The facts that CRK45 affects many genes up- and down-stream of the SA-dependent signaling pathway, such as ICS1, EDS5 and WRKYs (Figs. 4 and 6), forced us to suspect that CRK45 is not just well-controlled at the transcriptional level. As a protein kinase, regulation at the enzyme activity level should be as important as, or probably be more important than, that at the transcriptional level. One hypothesis we could assume currently is that after pathogen infection, elevated expression of the CRK45 transcript leads to the CRK45 protein synthesized de novo, and the accumulated CRK45 was then recruited by the

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Fig. 7. Expression of WRKY70 and CRK45 in wild type, the npr1, wrky70 and cpr5 mutants after Pst DC3000 treatment. Leaves of 4-week-old wild type, the npr1, wrky70 and cpr5 mutants were inoculated with 5  107 cfu mL1 of Pst DC3000. Samples were harvested at indicated times and total RNA was extracted for qRT-PCR analysis, Two independent experiments were performed and the results were similar.

membrane bound, DUF26 containing receptor-like kinase CRK36, as suggested by Tanaka et al., and formed a protein kinase complex to transduce signals further, just as that worked out for the BIK1FLS2-BAK1 complex in early flagellin signaling [18]. The activated CRK45 should further act on its target proteins and activate them, given by the phosphorylation process. And some of its targets might again be transcription factors, hence, the activated transcription factors bind to the promoters of ICS1, EDS5, WRKYS and others, and regulate their expression further. So far, how CRK45 was activated by CRK36 and what kind of substrates it has yet remains elusive. Future investigation is still required to uncover the detailed mechanism on how CRK45 executes its function in both disease resistance and early seedling establishment in Arabidopsis. In summary, as a receptor-like protein kinase, CRK45 plays an important role in the interaction between plant and pathogen, and shows its new face in enhancing disease resistance rather than just in early seedling establishment. Our results strongly suggested that in SA-dependent defense signaling pathway, CRK45 was positively regulated by NPR1 and WRKY70 at the transcriptional level.

4. Materials and methods 4.1. Plant growth conditions The Arabidopsis thaliana wild type, mutants and transgenic plants used throughout this work are in the Columbia ecotype (Col0) background. The T-DNA insertion allele of CRK45 (Salk-057538) was obtained from Arabidopsis Biological Resource Center (ABRC). Soil-grown A. thaliana plants were maintained at 22  C in a growth chamber with a 14-h light cycle (100 mE/m2 s1). Plants of different genotypes were grown under the same conditions, and seeds were collected at the same time. 4.2. RNA extraction and real-time PCR analysis Total RNA was extracted according to the manufactures’ instructions (Invitrogen) with Trizol reagent. After DNase I (Ambion Cat# AM2224) treatment, 2 mg of RNA was used for reverse transcription with (Takara, Dalian, China Cat# D2640A). Real-time RTPCR analysis was performed using SYBR Green Perfect mix (TaKaRa, Cat# DRR041A) on a LightCycler 480 system (Roche), with the program of 40 cycles under the following conditions: 95  C for 5 s, 60  C for 30 s, and 72  C for 15 s. The transcript of EF1a was used to normalize the samples. Relative gene expression was calculated using double DCt method, which gives fold of gene induction relative to its basal level before treatment (zero hour time point). All primers used are listed in Supplementary Table S1. 4.3. RNA gel blot analysis Total RNA was extracted using Trizol reagent according to the manufactures’ instructions (Invitrogen). Twelve micrograms of total RNA was fractionated in a 1% agarose gel containing formaldehyde and blotted onto a nylon membrane (Roche mannhein, Germany Cat# 1417240). The membrane was then hybridized with a DIG (Roche, Digoxigenin Cat# 11636090910) labeled probe. Hybridization and detection were performed according to manufacturer’s instructions (Roche mannhein, Germany Cat# 11796895001, Cat# 11096176001, Cat# 11093274910 and Cat# 11655884001). The primers used are listed in Supplementary Table S2. 4.4. Pathogen infection assay

Fig. 8. A working model of CRK45 in plant defense response. The black lines showed the results of previous studies, the red lines showed our results, and the dashed lines indicated the possible regulation mechanism of CRK45 at post-transcriptional level. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

P. syringae pv. tomato DC3000 (Pst DC3000) strain (from 80  C stock, streaked onto a plate) grown on King’s medium B agar plate (pepteose peptone 20 g, glycerol 10 mL, K2HPO4 1.5 g, and add ddH2O to bring the volume to 1 L, adjust pH to 7.2, then add 15 g

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agar) containing 50 mg mL1 rifampicin at 28  C for about 36 h. The bacterial suspension was prepared in sterile water. Pst DC3000 bacteria were re-suspended to a final OD600 of 0.05 for gene expression analysis and SA concentration assay, OD600 of 0.0005 for bacterial growth assay and OD600 of 0.1 for spray inoculation assay. For gene expression analysis and SA concentration assay the leaves from 4- to 5-week-old plants were injected with a needleless 1 mL syringe and leaves were harvested at indicated times. For in planta bacterial growth assay the leaves from 4- to 5-week-old plants were injected with a needleless 1 mL syringe and colony-forming units (cfu) were calculated at 0, 2 and 4 days after inoculation. For spray inoculation assay, the 4- to 5-week-old plants were spray the bacterial suspension and covered with plastic film for three days. 4.5. B. cinerea infection assay B. cinerea (Strain: DSM 4709) were grown on potato dextrose agar medium (Difco, Detroit, MI). Plates with 2.4% potato dextrose and 1.5% agar were used to maintain the B. cinerea culture. For harvesting spores, 1.2% potato dextrose and 1.5% agar were used. After 12e14 days, spores were harvested by flooding with halfstrength liquid MS medium. The medium containing fungal spores were centrifuged at 2900 g for 15 min. Spore pellets were suspended in liquid medium, and diluted to the required concentration. For gene expression analysis twelve- to fourteen-day-old seedlings grown in GC vials were inoculated with B. cinerea spores at a final concentration of 4.0  105 spores per vial and all plants in GC vials were harvested at indicated times. 4.6. Generation of transgenic plants For complementary of the crk45 mutant, a 1.5-kb fragment of the CRK45 coding region was amplified by PCR from Arabidopsis genomic DNA with the forward (50 -TAGGATCCATGGCCGTTACTTCGCTTCTC-30 ) and the reverse (50 -CAG-TCGACGCACTCAATCTCCTTAACGTGA-30 ) primers. The PCR product was ligated into the pENTR-D-TOPO vector following the manufacturer’s protocol (Invitrogen). After sequencing confirmation, the CRK45 gDNA was cloned into the Gateway-compatible pMDC32 [31] binary vector through the LR recombination reaction (Invitrogen) to obtain the CRK45 complementary construct. Than the constructs were transformed into Agrobacterium tumefaciens strain GV3101 and introduced into the crk45 mutant by the floral dip method. Transgenic seeds were screened on half-strength MS plates containing 0.8% (w/v) agar and 15 mg mL1 of hygromycin. The individual CRK45 complementary lines were checked by qRT-PCR analyses for CRK45 expression level. 4.7. SA and MeJA treatment For SA and MeJA treatment, 4-weed-old seedlings growing in soil were sprayed with 1 mM SA or MeJA. Samples were harvested at various time points. 4.8. SA measurement Total and free SA concentration was quantified according to our previous report [32]. Briefly, the 4-weed-old seedlings growing in soil were inoculated with Pst DC3000 (5  105 cfu/mL (OD600 ¼ 0.0005)). Samples were harvested at indicated times. The frozen leaf tissue (100 mg) was ground in liquid nitrogen, extracted with methanol twice and the supernatants from both extractions were combined and dried in a speedVac concentrator (Eppendorf). The dried samples were re-suspended in trichloroacetic acid (TCA).

Then partitioning twice with ethyl acetate:cyclohexane (1:1, v/v), the organic phase was combined and dried in a speedVac, and the residue was re-suspended in the mobile phase for free SA. The TCA phase was treated with hydrochloric acid at 80  C for 1 h, and then repeated steps of the free SA extraction to obtain the glucoside conjugate SA (SAG). Sample analysis was performed on a LC-20A series HPLC system (Shimadzu) using a Shim-pack C18 column (150  4.6 mm), with the mobile phase (methanol: 0.03 M sodium acetate, 20:80, v/v) flow rate of 0.8 mL min1. Fluorescent detection was performed on an HPLC spectrofluorescence detector at an excitation/emission wavelength of 300/410 nm. Acknowledgments We thank Dr. Yiji Xia for the crk45 and wrky70 mutants, Dr. Jianmin Zhou for providing the P. syringae strains, and Dr. Xinnian Dong for the npr1 and cpr5 mutants. This work was supported by the grants from National Natural Science Foundation and Ministry of Education of China (grant nos. 30860029 and NCET-11-1020 to R.W.), and from Ministry of Science and Technology of China and Inner Mongolia Agricultural University (grant nos. 2011AA100203 and NDPYTD2010-3 to G. L.) Appendix A. Supplementary data Supplementary data related to this article can be found at http:// dx.doi.org/10.1016/j.plaphy.2013.10.024. References [1] P.J. Rushton, I.E. Somssich, Transcriptional control of plant genes responsive to pathogens, Curr. Opin. Plant Biol. 1 (1998) 311e315. [2] J. Shah, The salicylic acid loop in plant defense, Curr. Opin. Plant Biol. 6 (2003) 365e371. [3] W.E. Durrant, X. Dong, Systemic acquired resistance, Annu. Rev. Phytopathol. 42 (2004) 185e209. [4] X. Dong, NPR1, all things considered, Curr. Opin. Plant Biol. 7 (2004) 547e552. [5] T. Eulgem, Dissecting the WRKY web of plant defense regulators, PLoS Pathog. 2 (2006) e126. [6] T. Eulgem, P.J. Rushton, S. Robatzek, I.E. Somssich, The WRKY superfamily of plant transcription factors, Trends Plant Sci. 5 (2000) 199e206. [7] S.P. Pandey, I.E. Somssich, The role of WRKY transcription factors in plant immunity, Plant Physiol. 150 (2009) 1648e1655. [8] I. Ciolkowski, D. Wanke, R.P. Birkenbihl, I.E. Somssich, Studies on DNA-binding selectivity of WRKY transcription factors lend structural clues into WRKYdomain function, Plant Mol. Biol. 68 (2008) 81e92. [9] M.C. van Verk, D. Pappaioannou, L. Neeleman, J.F. Bol, H.J. Linthorst, A novel WRKY transcription factor is required for induction of PR-1a gene expression by salicylic acid and bacterial elicitors, Plant Physiol. 146 (2008) 1983e1995. [10] J. Dong, C. Chen, Z. Chen, Expression profiles of the Arabidopsis WRKY gene superfamily during plant defense response, Plant Mol. Biol. 51 (2003) 21e37. [11] D. Wang, N. Amornsiripanitch, X. Dong, A genomic approach to identify regulatory nodes in the transcriptional network of systemic acquired resistance in plants, PLoS Pathog. 2 (2006) e123. [12] S.H. Shiu, A.B. Bleecker, Plant receptor-like kinase gene family: diversity, function, and signaling, Sci. STKE 2001 (2001) re22. [13] S.H. Shiu, A.B. Bleecker, Expansion of the receptor-like kinase/Pelle gene family and receptor-like proteins in Arabidopsis, Plant Physiol. 132 (2003) 530e543. [14] M. Wrzaczek, M. Brosche, J. Salojarvi, S. Kangasjarvi, N. Idanheimo, S. Mersmann, S. Robatzek, S. Karpinski, B. Karpinska, J. Kangasjarvi, Transcriptional regulation of the CRK/DUF26 group of receptor-like protein kinases by ozone and plant hormones in Arabidopsis, BMC Plant Biol. 10 (2010) 95. [15] P. Czernic, B. Visser, W. Sun, A. Savoure, L. Deslandes, Y. Marco, M. Van Montagu, N. Verbruggen, Characterization of an Arabidopsis thaliana receptorlike protein kinase gene activated by oxidative stress and pathogen attack, Plant J. 18 (1999) 321e327. [16] K. Chen, L. Du, Z. Chen, Sensitization of defense responses and activation of programmed cell death by a pathogen-induced receptor-like protein kinase in Arabidopsis, Plant Mol. Biol. 53 (2003) 61e74. [17] B.R. Acharya, S. Raina, S.B. Maqbool, G. Jagadeeswaran, S.L. Mosher, H.M. Appel, J.C. Schultz, D.F. Klessig, R. Raina, Overexpression of CRK13, an Arabidopsis cysteine-rich receptor-like kinase, results in enhanced resistance to Pseudomonas syringae, Plant J. 50 (2007) 488e499.

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