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First published online November 5, 2008; 10.1104/pp.108.130583 Plant Physiology 149:434-444 (2009) © 2009 American Society of Plant Biologists OPEN ACCESS ARTICLE
The Arabidopsis A4 Subfamily of Lectin Receptor Kinases Negatively Regulates Abscisic Acid Response in Seed Germination1,[W],[OA]Department of Biological Sciences, Lehman College, City University of New York, Bronx, New York 10468 (Z.X., G.Y., P.G., Z.-L.Z.); Cancer Center/Center for Immunobiology and Vaccine Development, Children's Hospital Oakland Research Institute, Oakland, California 94609 (A.W.); and Ph.D. Program in Biology, Graduate School and University Center, City University of New York, New York, New York 10016 (Z.-L.Z.)
Abscisic acid (ABA) is an important plant hormone for a wide array of growth and developmental processes and stress responses, but the mechanism of ABA signal perception on the plasma membrane remains to be dissected. A previous GeneChip analysis revealed that a member of the A4 subfamily of lectin receptor kinases (LecRKs) of Arabidopsis (Arabidopsis thaliana), At5g01540 (designated LecRKA4.1), is up-regulated in response to a low dose of ABA in the rop10-1 background. Here, we present functional evidence to support its role in ABA response. LecRKA4.1 is expressed in seeds and leaves but not in roots, and the protein is localized to the plasma membrane. A T-DNA knockout mutant, lecrka4.1-1, slightly enhanced ABA inhibition of seed germination. Interestingly, LecRKA4.1 is adjacent to two other members of the A4 subfamily of LecRK genes, At5g01550 (LecRKA4.2) and At5g01560 (LecRKA4.3). We found that loss-of-function mutants of LecRKA4.2 and LecRKA4.3 exhibited similarly weak enhancement of ABA response in seed germination inhibition. Furthermore, LecRKA4.2 suppression by RNA interference in lecrka4.1-1 showed stronger ABA inhibition of seed germination than lecrka4.1-1, while the response to gibberellic acid was not affected in lecrka4.1-1 and lecrka4.1-1; LecRKA4.2 (RNAi) lines. Expression studies, together with network-based analysis, suggest that LecRKA4.1 and LecRKA4.2 regulate some of the ABA-responsive genes. Taken together, our results demonstrate that the A4 subfamily of LecRKs has a redundant function in the negative regulation of ABA response in seed germination.
Abscisic acid (ABA) is a hormone that modulates a wide spectrum of growth and developmental processes (such as seed maturation, dormancy, and germination) and responses to biotic and abiotic stresses (Finkelstein et al., 2002  ; Himmelbach et al., 2003; Xie et al., 2005). Although ABA signal perception and transduction pathways have not been clearly demonstrated yet, nearly 50 genes have been shown to affect ABA responses through forward and reverse genetics (Finkelstein et al., 2002). Among them, the ABA signaling proteins that are associated with the plasma membrane (PM) are of particular interest (Wang and Zhang, 2008; Wasilewska et al., 2008; Zheng and Xin, 2008). It is well recognized that PM is an important site for ABA perception (Finkelstein et al., 2002; Yamazaki et al., 2003; Wang and Zhang, 2008; Zheng and Xin, 2008), although direct biochemical evidence has been reported that ABA can also be sensed in the plastid (Shen et al., 2006) or the nucleus (Razem et al., 2006).
Two major types of PM-associated proteins have been increasingly recognized as important ABA perception or signaling components. The first one is the PM-integral receptor-like kinases. Among more than 600 receptor-like kinases in Arabidopsis (Arabidopsis thaliana), RPK1 was shown to be induced starting 1 h after the ABA treatment (Hong et al., 1997
The second type of PM-associated ABA signaling proteins includes heterotrimeric GTP-binding proteins such as GPA1 and AGB1 (Wang et al., 2001
In an effort to understand the transcriptional control of ABA response by ROP10 small GTPase, a negative regulator of ABA signaling (Zheng et al., 2002
LecRKA4.1 Expression and Protein Subcellular Localization Patterns To determine where LecRKA4.1 is expressed, both reverse transcription (RT)-PCR and promoter:GUS reporter analyses were performed. RT-PCR analysis showed that LecRKA4.1 is preferentially expressed in the shoots of young seedlings, while its transcript is barely detectable in the roots (Fig. 1A ). LecRKA4.1 is also weakly expressed in the stems and flowers but strongly expressed in the leaves (Fig. 1A). Consistent with the RT-PCR results, a 1.2-kb fragment of LecRKA4.1 promoter directed GUS reporter expression in the shoots but not in the roots of young seedlings (Fig. 1, B and C). This indicates that the 1.2-kb promoter fragment likely contains all of necessary cis elements for proper expression of the LecRKA4.1 gene. Using three homozygous PLecRKA4.1:GUS lines, all with one T-DNA insertion, we found that the LecRKA4.1 promoter is very active in the vascular system and trichomes of the leaf (Fig. 1D), and it is also expressed in the guard cells (Fig. 1E). In addition, this promoter is active in anthers and stigmas but not in petals (Fig. 1F). We also found that the LecRKA4.1 promoter is active in germinating seeds, although its activity is absent in the protruding radicles (Fig. 1, G and H).
The intronless LecRKA4.1 gene has a translated region of 2,049 bp, encoding a protein of 682 amino acids. The predicted protein has an N-terminal signal peptide (amino acids 1–27), legume lectin  and β domains (amino acids 29–278), and a transmembrane domain (amino acids 310–332) that is followed by a protein kinase domain (amino acids 367–641) and the C terminus. To confirm that LecRKA4.1 is a PM-localized protein, we transiently expressed LecRKA4.1-GFP fusion protein in leaf epidermal cells of a transgenic line that expresses an mCherry version of red fluorescent protein (RFP). Compared with RFP, which is localized to the cytoplasm and the nucleus, LecRKA4.1-GFP is exclusively localized to the PM (Fig. 1I). Because the N-terminal half of LecRKA4.1 contains the signal peptide, lectin domains, and a transmembrane domain, we constructed a GFP fusion with the C-terminal half of LecRKA4.1, LecRKA4.1351–682, which contains the predicted protein kinase domain. Transient expression in leaf epidermal cells showed that LecRKA4.1351–682-GFP was expressed in the nucleus and the cytoplasm (Fig. 1I). This pattern is similar to that of the RFP control, as shown in the merged RFP and GFP images. Therefore, we conclude that the transmembrane domain-containing N-terminal half of LecRKA4.1 is responsible for targeting LecRKA4.1 specifically to the PM.
To demonstrate that the PM-localized LecRKA4.1 has a function in ABA response, we isolated and characterized a T-DNA line of LecRKA4.1 (SALK_ 070801), which is designated lecrka4.1-1. RT-PCR analysis showed that LecRKA4.1 transcript was absent in lecrka4.1-1 (Fig. 2A ). Given that the T-DNA is located in the exon (1,434 bp from ATG), we believe that lecrka4.1-1 likely represents a null allele of LecRKA4.1. LecRKA4.1 is not expressed in the roots (Fig. 1A), and indeed, root growth inhibition response to ABA was not significantly altered in lecrka4.1-1 (data not shown). In addition, we did not observe any difference in leaf water loss in lecrka4.1-1 (data not shown), although the LecRKA4.1 promoter is active in the guard cells (Fig. 1E). Therefore, we focused our ABA phenotypic characterization on seed germination inhibition. After 3 d on the medium containing 0.6 µM ABA, the Columbia (Col) wild type had a germination percentage of 60%, but the germination percentage of lecrka4.1-1 was only 27% (Fig. 2B). This 55% reduction of seed germination percentage in lecrka4.1-1 indicates that the LecRKA4.1 mutant slightly increased the ABA sensitivity of seed germination inhibition. To substantiate that this enhancement is due to the loss of LecRKA4.1 function, a functional complementation test was performed. The full-length LecRKA4.1 cDNA under the control of a 2X35S cauliflower mosaic virus (CaMV) promoter was transformed into lecrka4.1-1, and two independently transformed lines, 7-2 and 11-4, had high levels of LecRKA4.1 transcript (Fig. 2A). In the absence of ABA, these two lines had almost 100% germination, similar to the wild type and lecrka4.1-1, and under 0.6 µM ABA, they had seed germination percentages close to that for the wild type compared with reduced germination for lecrka4.1-1 (Fig. 2B). These results suggest that LecRKA4.1 is a negative regulator of ABA response in seed germination.
Mutants of LecRKA4.2 and LecRKA4.3 Slightly Enhanced ABA Response in Seed Germination Inhibition The weak phenotype of lecrka4.1-1 indicates that other members of the A4 subfamily of LecRKs could have redundant functions. Interestingly, two other members, At5g01550 (designated LecRKA4.2) and At5g01560 (LecRKA4.3), are immediately adjacent to LecRKA4.1 (At5g01540), and they share very high amino acid sequence identity (65%–74%). Therefore, we isolated and characterized their T-DNA insertional lines. In the Salk T-DNA database, there was one line for LecRKA4.2 (SALK_108000, designated lecrka4.2-1) and two lines for LecRKA4.3, SALK_003084 (designated lecrka4.3-1) and SALK_122197 (designated lecrka4.3-2). RT-PCR analysis showed that LecRKA4.2 transcript was lower than that in the wild type and that LecRKA4.3 was absent in the lecrka4.3-1 and lecrka4.3-2 alleles (Fig. 3A ). As in the case of lecrka4.1-1, the T-DNA insertion in lecrka4.2-1, lecrka4.3-1, and lecrka4.3-2 did not interfere with the expression of other A4 subfamily genes.
We then subjected the mutants to 0.3 µM ABA treatment. The germination kinetic profile showed that, similar to lecrka4.1-1, fewer lecrka4.2-1, lecrka4.3-1, and lecrka4.3-2 seeds germinated than wild-type seeds at 1.5 d after cold treatment, and after 3 d, they all germinated like wild-type seeds (Fig. 3B). At day 2, lecrka4.3-2 still germinated less, while lecrka4.3-1 had a similar germination percentage as the wild type. The reason for this allelic difference is unknown. A dose-response curve showed that after 4 d at both 0.6 and 0.9 µM ABA, the mutants for all three genes had lower germination rates than the wild type (Fig. 3C). These experiments indicate that the A4 subfamily of LecRKs encoded by the three genes clustered on chromosome 5 probably has a redundant function in ABA-mediated seed germination inhibition response.
If LecRKA4.1, LecRKA4.2, and LecRKA4.3 have redundant functions as negative regulators of ABA response, we would expect to see a stronger phenotype in their double and triple mutants. However, their extremely tight genetic linkage prevented us from obtaining double or triple mutants by crossing with each other. Therefore, we decided to use RNA interference (RNAi) to suppress LecRKA4.2 and LecRKA4.3 gene expression in the lecrka4.1-1 background. Given their high sequence homology (77% identical for full-length cDNAs), we initially predicted that the LecRKA4.2 RNAi using either the partial fragment of LecRKA4.2 cDNA (1,130–1,760 bp) or the longer cDNA fragment (1–1,760 bp) would suppress both LecRKA4.2 and LecRKA4.3 expression. Two representative RNAi lines, S2 and F9, were chosen for further characterization. LecRKA4.2 mRNA expression was greatly suppressed in S2 and F9, but LecRKA4.3 expression was not altered (Fig. 4A ).
Seed germination kinetic profiles for the wild type, lecrka4.1-1, and the two RNAi lines, S2 and F9, at 0.3 µM ABA showed that after 1.5 d, while lecrka4.1-1 had a germination percentage about 50% lower than the wild type, S2 and F9 lines were even 50% to 80% lower than lecrka4.1-1 (Fig. 4B). After 2 d, although lecrka4.1-1 had a similar germination percentage as the wild type (approximately 82%), only 48% to 64% of seeds in S2 and F9 lines germinated. Furthermore, after 7 d, almost all of the wild-type and lecrka4.1-1 seeds germinated, but 14% to 19% of the seeds in S2 and F9 still did not germinate. On the other hand, an ABA dose-response curve showed that after 4 d, S2 and F9 lines had lower seed germination percentages than lecrka4.1-1 (Fig. 4C). We also examined the postgerminative seedling growth by scoring the cotyledon-greening phenotype, and the results showed that S2 and F9 had 50% fewer seedlings showing green cotyledons than lecrka4.1-1 after 6 d in the presence of 0.6 µM ABA (Fig. 4D) or after 9 d in the presence of 0.9 µM ABA (Fig. 4E). Taken together, these results show that suppression of LecRKA4.2 expression further enhances the ABA sensitivity observed in lecrka4.1-1. To exclude the possibility that the enhanced response to ABA in lecrka4.1-1 and lecrka4.1-1; LecRKA4.2 (RNAi) lines is due to differences in seed germination or postgerminative seedling growth that are not related to ABA signaling, freshly harvested seeds of these mutant lines were allowed to germinate in the absence of ABA. The results showed that lecrka4.1-1 and the S2 and F9 lines exhibited a very similar germination kinetic profile (Supplemental Fig. S1). Furthermore, treatments with 1 and 10 µM GA3, which antagonizes the ABA inhibitory effect in seed germination, did not result in lower sensitivity to GA3 in lecrka4.1-1 and the S2 and F9 lines (Supplemental Fig. S2). These results support the idea that the observed seed germination response in the LecRKA4 subfamily gene knockout or RNAi lines is likely specific to ABA.
To test whether ABA-responsive genes are negatively regulated by the A4 subfamily LecRK, expression of ABI1 and ABI2 genes was quantified by real-time RT-PCR. Consistent with a previous report (Leung et al., 1997
To further explore which genes are possibly regulated by the three members of A4 subfamily LecRK or their pathway, a protein-protein interaction network was assembled. The protein-protein interactions were based on both experimentally demonstrated or predicted physical interactions (Geisler-Lee et al., 2007 ) and functional interactions implicated by a large number of gene expression analyses (Ma et al., 2007). We found that LecRKA4.1 and LecRKA4.2, but not LecRKA4.3, were present in the network database, resulting in a total of 70 unique genes (nodes; Supplemental Table S1) that exhibit 149 physical or functional interactions (edges). Therefore, a network for LecRKA4.1 and LecRKA4.2 was assembled (Fig. 6
). Functional categorical analysis of these 70 genes showed that genes involved in metabolism (in particular phosphate metabolism), protein fate (folding, modification, and destination), cellular transport, interaction with environments, and cell rescue, defense, and virulence were overly represented compared with the whole genome representation (Supplemental Table S1).
We then selected two transcription factor genes from the assembled network for quantitative analysis of their expression in lecrka4.1-1 and the F9 line. AZF1, although not yet updated in the database as ABA suppressed, was recently shown to be down-regulated by ABA (Sakamoto et al., 2004 ), and its close homolog, AZF2, was demonstrated to act as a transcription factor negatively regulating ABA response (Sakamoto et al., 2004). Consistently, we found that AZF1 mRNA level was reduced by about 75% and 90% by 0.3 and 0.9 µM ABA, respectively (Fig. 5D). Surprisingly, although AZF1 showed a direct functional interaction with LecRKA4.1 based on the assembled network (Fig. 6), there was no difference in its transcript levels between the wild type and lecrka4.1-1 at 0, 0.3, and 0.9 µM ABA (Fig. 5D). However, F9 had a 50% lower expression level than the wild type in the absence of ABA and a 65% lower level at 0.3 µM ABA. At 0.9 µM ABA, a similar trend was observed. In contrast to AZF1, WRKY53, another transcription factor gene that was down-regulated by ABA, as reported by the Genevestigator Web site (https://www.genevestigator.ethz.ch/gv/index.jsp) and in our experimental conditions (Fig. 5E), showed differential expression levels between the wild type, lecrka4.1-1, and F9 (Fig. 5E). Although there was no statistical difference between lecrka4.1-1 and the wild type in the no-ABA control, the 50% lower level difference in lecrka4.1-1 compared with the wild type at 0.3 µM ABA was statistically significant. Interestingly, as in the case of At5g27420 under the 0.3 µM ABA treatment (Fig. 5C), F9 showed a further reduction in WRKY53 expression compared with lecrka4.1-1 (Fig. 5E). In the control, there was an approximately 2-fold difference between F9 and the wild type, but at 0.3 µM ABA, they showed a 5-fold difference. At 0.9 µM ABA, there was still a 3-fold difference between F9 and the wild type.
In this work, we provide genetic evidence that LecRKA4.1 and the other two most closely related LecRKs (LecRKA4.2 and LecRKA4.3) act as negative regulators of ABA response in seed germination. First, we show that single knockout mutants for each of these three genes exhibit subtle enhancements in ABA-inhibited seed germination. Second, down-regulation of LecRKA4.2 by RNAi further enhanced the response in lecrka4.1-1. Third, this enhancement is not due to the developmental delay in lecrka4.1-1 and lecrka4.1-1; LecRKA4.2 (RNAi) lines or to their altered sensitivity to GA3. Fourth, gene expression studies, coupled with gene network assembly, suggest that LecRKA4.1 and LecRKA4.2 control the expression of some ABA-responsive genes. Therefore, our results demonstrate that the A4 subfamily of LecRKs acts redundantly in the negative regulation of ABA response.
The identification of the PM-associated ABA signaling components, including ABA receptors, has been a significant challenge. Some of the components, such as RPK1 (Hong et al., 1997
However, it is difficult to elucidate how the A4 subfamily of LecRKs links to ROP10 small GTPase in ABA signaling. The observation that LecRKA4.1 was up-regulated by a low dose (1 µM) but not at higher doses (10 and 100 µM) of ABA in rop10-1 seedlings indicates that LecRKA4.1 might participate in the ROP10-gated, low-dose ABA response pathway (Xin et al., 2005
Nevertheless, the identification of three members of the A4 subfamily of LecRKs as redundant, negative regulators of ABA response will provide novel insights into the complex ABA signaling networks. Recently, plastidic and nuclear ABA receptors have been found (Razem et al., 2006
The role of the legume lectin-like extracellular domain in ABA signaling is intriguing. Lectins are known to bind to carbohydrates, and some of them have been shown to act as defense proteins or in plant development (Herve et al., 1996 Another interesting question that needs to be addressed concerns the pathways controlled by these LecRKs. Consistent with their weak ABA response phenotypes, the mutants and transgenic lines exhibit slight differences in ABI1/ABI2 expression from their wild types, indicating that these LecRKs may not control a major ABA response pathway but only modulate a minor branch of the pathway. Although more dramatic differences might be observed if three members of the A4 subfamily of LecRK genes are knocked out, currently this possibility cannot be tested because they physically cluster on the same chromosomal location. Furthermore, the assembled network and gene expression studies indicate that LecRKA4.1 and LecRKA4.2 may have overlapping and distinct response pathways. For example, while the mutation in LecRKA4.1 increased ABI1 and ABI2 expression, LecRKA4.2 (RNAi) did not further enhance their expression (Fig. 5, A and B). Similarly, it is less likely that AZF1 was predominantly affected by LecRKA4.1, but a reduction in LecRKA4.2 expression could enhance the ABA suppression of AZF1 expression (Fig. 5D). Nevertheless, down-regulation of the genes At5g27420 (encoding a zinc finger family protein) and WRKY53 (a transcription factor) was enhanced by the loss of LecRKA4.1 and LecRKA4.2 function (Fig. 5, C and E). Regardless of whether or not these LecRKs modulate a minor ABA signaling branch through overlapping or distinct pathways, it is important to first identify their downstream factors in ABA signaling in the future. Therefore, genetic and biochemical analyses of those components on the assembled network will provide further insights into the complex ABA signaling networks.
Plant Materials, T-DNA Mutant Isolation, and Growth Conditions The lecrka4.1-1 (SALK_ 070801), lecrka4.2-1 (SALK_108000), lecrka4.3-1 (SALK_003084), and lecrka4.3-2 (SALK_122197) mutants of Arabidopsis (Arabidopsis thaliana) were isolated from T-DNA insertion lines maintained in the Arabidopsis Biological Resource Center at The Ohio State University. For screening homozygous mutants, the following primers were used: XZP136 (sense, 5'-CATCCTCGCAGATTGAGATACAG-3') and XZP60 (antisense, 5'-TCGACGAAACATATCCTACCAACT-3') for lecrka4.1-1; XZP211 (sense, 5'-GTTTTCAAAGGGGAAGAACATACG-3') and XZP212 (antisense, 5'-GGGTTGGAGATAGCGTGAAGGTG-3') for lecrka4.2-1; and XZP213 (sense, 5'-CTCTGTCTTCTTTGCTTCTTCATCTCG-3') and XZP214 (antisense, 5'-CGGCTATAAAGAAGGTCCCAGAG-3') for lecrka4.3-1 and lecrka4.3-2. Seeds of the Arabidopsis wild type (Col), T-DNA insertion mutants, and transgenic plants were cold treated at 4°C for 3 to 4 d and then germinated and grown in the greenhouse for harvesting seeds, or in an incubator at 22°C with a 16-h-light/8-h-dark cycle for seed germination assay.
Seed germination was performed on half-strength Murashige and Skoog (MS) medium supplemented with various ABA concentrations. Petri plates were incubated at 22°C in the light after cold treatment (4°C for 3–4 d). Germination was scored every 12 h after incubation in the light, with the criterion being set as the complete protrusion of the radicle. For cotyledon-greening characterization, seedlings were observed from 5 to 9 d after cold treatment. For the seed germination assay with GA3, seeds were plated on half-strength MS medium supplemented with various GA3 concentrations. After 3 d of cold treatment, germination was scored every 2 h after incubation in the light, with the same germination criterion used above.
For regular PCR analysis of LecRK gene expression, 7-d-old young seedlings were used for total RNA using TRIzol (Invitrogen). A total of 5 µg of RNA was reverse transcribed in a 20-µL reaction using SuperScript III reverse transcriptase and oligo(dT)12–18 primer (Invitrogen) according to the instructions provided by the vender. PCR was performed using Taq DNA polymerase, with ACT2 as the internal control, using the primers ACT2S and ACT2A described previously (Xin et al., 2005
For real-time PCR analysis of gene expression, seeds of lecrka4.1-1, its wide type (Col), and a lecrka4.1-1; LecRKA4.2 (RNAi) line (F9) were incubated in 50 mL of liquid MS medium, pH 5.7, at 4°C for 3 d and then allowed to germinate and grow at 22°C for 4 d under a 16-h-day/8-h-night regime with continuous shaking at 150 rpm. After three rinses with freshly prepared MS liquid medium, young seedlings were treated with 0, 0.3, and 0.9 µM ABA that was dissolved with dimethyl sulfoxide (with the final concentration in the medium of 0.1%, v/v) and prepared in the MS medium. After 12 h of treatment, the seedlings were immediately frozen in liquid nitrogen and stored at –80°C until RNA isolation. Three biological replicates were performed at different times for all genotype/treatment combinations. RNA RT was described above, and real-time quantitative PCR analysis was performed using the QuantiTect SYBR Green PCR kit (Qiagen). Real-time PCR was carried out in the MasterCycler II (Cypheid) according to the manufacturer's protocol. The primers for ACT2 were designed previously (Zheng et al., 2002
The LecRKA4.1 full-length cDNA was amplified using primers XZP147 (sense, 5'-ACTGCAGATGGGCACACAAAGATCCATG-3') and XZP148 (antisense, 5'-GCAGATCTCTACTGACTGATACGAGAAGTC-3'), with the underlined bases indicating the introduced restriction enzyme sites for cloning. The PCR product was digested by PstI and BglII and then cloned into GZ42, which is based on pCAMBIA1301 (B4), containing a hygromycin resistance selection marker and a 2X35S promoter of CaMV.
The LecRKA4.1 promoter fragment containing a 1.2-kb region upstream of the ATG codon and 20 bp of coding DNA downstream of ATG was amplified with primers XZP199 (sense, 5'-GGCGTCGACCTCAGTCCCATCTTTCTC-3') and XZP200 (antisense, 5'-CATGGATCCTTGTGTGCCCATGGGTG-3') using TaKaRa ExTaq (TaKaRa Bio). Underlined bases show the restriction enzyme sites for cloning. The amplified DNA fragment was digested by SalI and NcoI and then cloned into SalI and NcoI sites of the binary vector pCAMBIA1301 (B4) that contains GUS and the CaMV 35S terminator. The resulting plasmid, XZ22, was then transformed into Col using the floral dip method (Clough and Bent, 1998
LecRKA4.1 full-length cDNA was amplified with XZP147 (sense, as described above) and XZP198 (antisense, 5'-CATCTAGACTGACTGATACGAGAAGTCGAAGAAAC-3'), then digested by PstI and XbaI and cloned into the pCAMBIA3301-based binary vector GZ6 that contains GFP and the CaMV 35S promoter and terminator, resulting in the 35S:LecRKA4.1-GFP plasmid, designated XZ16. A cDNA fragment (1,051–1,339 bp) of LecRKA4.1 was amplified with XZP196 (sense, as described above) and XZP193 (antisense, 5'-CTAAGCTTCCGTTGGGGATGTAATCG-3') and then digested by PstI and HindIII. This fragment was cloned into XZ16 (digested by PstI and HindIII), giving rise to the LecRKA4.1351–682-GFP plasmid, designated XZ25. XZ16 and XZ25 were bombarded into young leaves of 35S:mCherry (RFP) lines, according to the instructions with the Bio-Rad Biolistic PDS-1000/He Particle Delivery System. The 35S:mCherry plasmid was constructed by replacing GFP in GZ6 (described above) with a cDNA fragment encoding the mCherry version of RFP (Shaner et al., 2004
The LecRKA4.2 cDNA fragment (1–1,760 bp) was amplified with primers XZP206 (sense, 5'-TTCTTAATTAACCATGGATGCTCGTGCTCTTCTTGCT-3') and XZP205 (antisense, 5'-AAGGATCCATTTAAATGAATCTCTCCTCTCGCGTGCAAT-3') using TaKaRa ExTaq (TaKaRa Bio). The amplified fragment was digested by SwaI and NcoI and then cloned into the pFGC5941 double-stranded RNA vector. Subsequently, this amplified fragment was digested by BamHI and PacI and cloned into the above resulting plasmid, giving rise to the RNAi plasmid, designated XZ20. Another RNAi plasmid, XZ21, was similarly obtained by amplifying the LecRKA4.2 cDNA fragment (1,130–1,760 bp) with primers XZP204 (sense, 5'-AATCTAGACCATGGTCCGAGGAAACTTATCGTCACC-3') and XZP205 (antisense, as described above). This fragment was then digested by SwaI-NcoI and BamHI-XbaI and sequentially cloned into pFGC5941. XZ20 and XZ21 were then transformed into the lecrka4.1-1 knockout mutant using the floral dip method (Clough and Bent, 1998
We assembled a protein-protein interaction network using the network assembly methods as adopted previously (Chuang et al., 2007 Sequence data from this article can be found in the GenBank/EMBL data libraries under accession numbers At5g01540 (LecRKA4.1), At5g01550 (LecRKA4.2), and At5g01560 (LecRKA4.3).
The following materials are available in the online version of this article.
We are grateful to Dr. Bo Liu (University of California, Davis) for providing the mCherry version of RFP, with permission for use from Dr. Roger Y. Tsien (University of California, San Diego). We thank Dr. Matthew Geisler (Southern Illinois University) for his advice on the Arabidopsis protein-protein interaction database. We also thank the Arabidopsis Biological Resource Center at The Ohio State University for providing various T-DNA lines. We appreciate the constructive and helpful comments on the manuscript from the editor and the reviewers. Received September 30, 2008; accepted November 2, 2008; published November 5, 2008.
1 This work was supported by the U.S. Department of Agriculture National Research Initiative Competitive Grants Program (grant no. 2004–35304–14911 to Z.-L.Z.).  The author responsible for distribution of materials integral to the findings presented in this article in accordance with the policy described in the Instructions for Authors (www.plantphysiol.org) is: Zhi-Liang Zheng (zhiliang.zheng{at}lehman.cuny.edu).
[W] The online version of this article contains Web-only data.
[OA] Open Access articles can be viewed online without a subscription. www.plantphysiol.org/cgi/doi/10.1104/pp.108.130583 * Corresponding author; e-mail zhiliang.zheng{at}lehman.cuny.edu.
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