CDPK5 and CDPK13 play key roles in acclimation to low oxygen through the control of RBOH-mediated ROS production in rice.

CALCIUM-DEPENDENT PROTEIN KINASE (CDPK) stimulates reactive oxygen species (ROS)-dependent signaling by activating RESPIRATORY BURST OXIDASE HOMOLOG (RBOH). The lysigenous aerenchyma is a gas space created by cortical cell death that facilitates oxygen diffusion from the shoot to the root tips. Previously, we showed that RBOHH is indispensable for the induction of aerenchyma formation in rice (Oryza sativa) roots under low-oxygen conditions. Here, we showed that CDPK5 and CDPK13 localize to the plasma membrane where RBOHH functions. Mutation analysis of the serine at residues 92 and 107 of RBOHH revealed that these residues are required for CDPK5- and CDPK13-mediated activation of ROS production. The requirement of Ca2+ for CDPK5 and CDPK13 function was confirmed using in vitro kinase assays. CRISPR/Cas9-based mutagenesis of CDPK5 and/or CDPK13 revealed that the double knockout almost completely suppressed inducible aerenchyma formation, whereas the effects were limited in the single knockout of either CDPK5 or CDPK13. Interestingly, the double knockout almost suppressed the induction of adventitious root formation, which is widely conserved in vascular plants, under low-oxygen conditions. Our results suggest that CDPKs are essential for the acclimation of rice to low-oxygen conditions, and also for many other plant species conserving CDPK-targeted phosphorylation sites in RBOH homologues.

NADPH oxidase-mediated sulfenylation of cysteine derivatives is key regulatory events for plant immune responses.

Reactive oxygen species (ROS) are rapidly generated during plant immune responses by RBOH, which is a plasma membrane-localizing NADPH oxidase. Although regulatory mechanisms of RBOH activity have been well documented, the ROS-mediated downstream signaling is unclear. We here demonstrated that ROS sensor proteins play a central role in the ROS signaling via oxidative post-translational modification of cysteine residues, sulfenylation. To detect protein sulfenylation, we used dimedone, which specifically and irreversibly binds to sulfenylated proteins. The sulfenylated proteins were labeled by dimedone in Nicotiana benthamiana leaves, and the conjugates were detected by immunoblot analyses. In addition, a reductant dissociated H2O2-induced conjugates, suggesting that cysteine persulfide and/or polysulfides are involved in sulfenylation. These sulfenylated proteins were continuously increased during both PTI and ETI in a RBOH-dependent manner. Pharmacological inhibition of ROS sensor proteins by dimedone perturbated cell death, ROS accumulation induced by INF1 and MEK2DD, and defense against fungal pathogens. On the other hand, Rpi-blb2-mediated ETI responses were rather enhanced by dimedone. These results suggest that the sulfenylation of cysteine and its derivatives in various ROS sensor proteins are important events in downstream of RBOH-dependent ROS burst to regulate plant immune responses.

Role of salicylic acid glucosyltransferase in balancing growth and defence for optimum plant fitness.

Salicylic acid (SA), an essential secondary messenger for plant defence responses, plays a role in maintaining a balance (trade-off) between plant growth and resistance induction, but the detailed mechanism has not been explored. Because the SA mimic benzothiadiazole (BTH) is a more stable inducer of plant defence than SA after exogenous application, we analysed expression profiles of defence genes after BTH treatment to better understand SA-mediated immune induction. Transcript levels of the salicylic acid glucosyltransferase (SAGT) gene were significantly lower in BTH-treated Nicotiana tabacum (Nt) plants than in SA-treated Nt control plants, suggesting that SAGT may play an important role in SA-related host defence responses. Treatment with BTH followed by SA suppressed SAGT transcription, indicating that the inhibitory effect of BTH is not reversible. In addition, in BTH-treated Nt and Nicotiana benthamiana (Nb) plants, an early high accumulation of SA and SA 2-O-ß-d-glucoside was only transient compared to the control. This observation agreed well with the finding that SAGT-overexpressing (OE) Nb lines contained less SA and jasmonic acid (JA) than in the Nb plants. When inoculated with a virus, the OE Nb plants showed more severe symptoms and accumulated higher levels of virus, while resistance increased in SAGT-silenced (IR) Nb plants. In addition, the IR plants restricted bacterial spread to the inoculated leaves. After the BTH treatment, OE Nb plants were slightly larger than the Nb plants. These results together indicate that SAGT has a pivotal role in the balance between plant growth and SA/JA-mediated defence for optimum plant fitness.

Retracted: "CITRX thioredoxin interacts with the tomato Cf-9 resistance protein and negatively regulates defence".

The authors regret to announce they would like to withdraw this paper, for two main reasons: Since the paper was published, it has become clear that the thioredoxin that interacts in yeast 2-hybrid with the Cf-9 C-terminus is in fact localized in the chloroplast, rendering a role in Cf-9 signalling unlikely. Close scrutiny of the figures suggests several duplications. - In Fig 3A, the Anti-MBP band in lane 4 closely resembles the antiMBP band in Fig 3B lane 1, though slightly rotated. - In Fig 6A, the leaf disc in the panel labelled TRV:00, -Avr9, 30 min looks identical to the leaf disc in Fig S5, panel labelled Cf2 TRV:CITRX, -Avr2, 1 h. - In Fig 6C, multiple bands appear duplicated. For example, GlucA, TRV:00, -Avr9, 0 h duplicated with 6 h; and GlucB, TRV:00, +Avr9, 0 h duplicated with Hin1, TRV:00, +Avr9, 0 h. Source data for these figures are not available. All the authors agree that this paper should be withdrawn from the scientific literature.

RNAi of the sesquiterpene cyclase gene for phytoalexin production impairs pre- and post-invasive resistance to potato blight pathogens.

Potato antimicrobial sesquiterpenoid phytoalexins lubimin and rishitin have been implicated in resistance to the late blight pathogen, Phytophthora infestans and early blight pathogen, Alternaria solani. We generated transgenic potato plants in which sesquiterpene cyclase, a key enzyme for production of lubimin and rishitin, is compromised by RNAi to investigate the role of phytoalexins in potato defence. The transgenic tubers were deficient in phytoalexins and exhibited reduced post-invasive resistance to an avirulent isolate of P. infestans, resulting in successful infection of the first attacked cells without induction of cell death. However, cell death was observed in the subsequently penetrated cells. Although we failed to detect phytoalexins and antifungal activity in the extract from wild-type leaves, post-invasive resistance to avirulent P. infestans was reduced in transgenic leaves. On the other hand, A. solani frequently penetrated epidermal cells of transgenic leaves and caused severe disease symptoms presumably from a deficiency in unidentified antifungal compounds. The contribution of antimicrobial components to resistance to penetration and later colonization may vary depending on the pathogen species, suggesting that sesquiterpene cyclase-mediated compounds participate in pre-invasive resistance to necrotrophic pathogen A. solani and post-invasive resistance to hemibiotrophic pathogen P. infestans.

An NADPH Oxidase RBOH Functions in Rice Roots during Lysigenous Aerenchyma Formation under Oxygen-Deficient Conditions.

Reactive oxygen species (ROS) produced by the NADPH oxidase, respiratory burst oxidase homolog (RBOH), trigger signal transduction in diverse biological processes in plants. However, the functions of RBOH homologs in rice (Oryza sativa) and other gramineous plants are poorly understood. Ethylene induces the formation of lysigenous aerenchyma, which consists of internal gas spaces created by programmed cell death of cortical cells, in roots of gramineous plants under oxygen-deficient conditions. Here, we report that, in rice, one RBOH isoform (RBOHH) has a role in ethylene-induced aerenchyma formation in roots. Induction of RBOHH expression under oxygen-deficient conditions was greater in cortical cells than in cells of other root tissues. In addition, genes encoding group I calcium-dependent protein kinases (CDPK5 and CDPK13) were strongly expressed in root cortical cells. Coexpression of RBOHH with CDPK5 or CDPK13 induced ROS production in Nicotiana benthamiana leaves. Inhibitors of RBOH activity or cytosolic calcium influx suppressed ethylene-induced aerenchyma formation. Moreover, knockout of RBOHH by CRISPR/Cas9 reduced ROS accumulation and inducible aerenchyma formation in rice roots. These results suggest that RBOHH-mediated ROS production, which is stimulated by CDPK5 and/or CDPK13, is essential for ethylene-induced aerenchyma formation in rice roots under oxygen-deficient conditions.

Nicotiana benthamiana MAPK-WRKY pathway confers resistance to a necrotrophic pathogen Botrytis cinerea.

MEK2-SIPK/WIPK cascade, a Nicotiana benthamiana mitogen-activated protein kinase (MAPK) cascade, is an essential signaling pathway for plant immunity and involved in hypersensitive response (HR) accompanied by cell death. WRKY transcription factors as substrates of SIPK and WIPK have been isolated and implicated in HR cell death. Here, we show virus-induced gene silencing of WRKY genes compromised constitutively active MEK2-triggered cell death in N. benthamiana leaves. In general, HR cell death enhances susceptibility to necrotrophic pathogens such as Botrytis cinerea. However, the WRKY gene silencing elevated susceptibility to B. cinerea. These findings suggest that downstream WRKYs of MEK2-SIPK/WIPK cascade are required for cell death-dependent and -independent immunities in N. benthamiana.

WRKY Transcription Factors Phosphorylated by MAPK Regulate a Plant Immune NADPH Oxidase in Nicotiana benthamiana.

Pathogen attack sequentially confers pattern-triggered immunity (PTI) and effector-triggered immunity (ETI) after sensing of pathogen patterns and effectors by plant immune receptors, respectively. Reactive oxygen species (ROS) play pivotal roles in PTI and ETI as signaling molecules. Nicotiana benthamiana RBOHB, an NADPH oxidase, is responsible for both the transient PTI ROS burst and the robust ETI ROS burst. Here, we show that RBOHB transactivation mediated by MAPK contributes to R3a/AVR3a-triggered ETI (AVR3a-ETI) ROS burst. RBOHB is markedly induced during the ETI and INF1-triggered PTI (INF1-PTI), but not flg22-tiggered PTI (flg22-PTI). We found that the RBOHB promoter contains a functional W-box in the R3a/AVR3a and INF1 signal-responsive cis-element. Ectopic expression of four phospho-mimicking mutants of WRKY transcription factors, which are MAPK substrates, induced RBOHB, and yeast one-hybrid analysis indicated that these mutants bind to the cis-element. Chromatin immunoprecipitation assays indicated direct binding of the WRKY to the cis-element in plants. Silencing of multiple WRKY genes compromised the upregulation of RBOHB, resulting in impairment of AVR3a-ETI and INF1-PTI ROS bursts, but not the flg22-PTI ROS burst. These results suggest that the MAPK-WRKY pathway is required for AVR3a-ETI and INF1-PTI ROS bursts by activation of RBOHB.

Molecular Characterization of LjABCG1, an ATP-Binding Cassette Protein in Lotus japonicus.

LjABCG1, a full-size ABCG subfamily of ATP-binding cassette proteins of a model legume, Lotus japonicus, was reported as a gene highly expressed during the early stages of nodulation, but have not been characterized in detail. In this study we showed that the induction of LjABCG1 expression was remarkable by methyl jasmonate treatment, and reporter gene experiments indicated that LjABCG1 was strongly expressed in the nodule parenchyma and cell layers adjacent to the root vascular tissue toward the nodule. LjABCG1 was suggested to be localized at the plasma membrane based on the fractionation of microsomal membranes as well as separation via aqueous two-phase partitioning. The physiological functions of LjABCG1 in symbiosis and pathogenesis were analyzed in homologous and heterologous systems. LjABCG1 knock-down L. japonicus plants did not show clear phenotypic differences in nodule formation, and not in defense against Pseudomonas syringae, either. In contrast, when LjABCG1 was expressed in the Arabidopsis pdr8-1 mutant, the penetration frequency of Phytophthora infestans, a potato late blight pathogen, was significantly reduced in LjABCG1/pdr8-1 than in pdr8-1 plants. This finding indicated that LjABCG1, at least partially, complemented the phenotype of pdr8 in Arabidopsis, suggesting the multiple roles of this protein in plant-microbe interactions.

Cell death-inducing stresses are required for defense activation in DS1-phosphatidic acid phosphatase-silenced Nicotiana benthamiana.

We previously identified DS1 plants that showed resistance to compatible Ralstonia solanacearum with accelerated defense responses. Here, we describe activation mechanisms of defense responses in DS1 plants. After inoculation with incompatible R. solanacearum 8107, DS1 plants showed hyperinduction of hypersensitive response (HR) and reactive oxygen species (ROS) generation. Transient expression of PopP1 and AvrA induced hyperinduction of HR and ROS generation. Furthermore, Pseudomonas cichorii (Pc) and a type III secretion system (TTSS)-deficient mutant of P. cichorii showed accelerated induction of HR and ROS generation. Chitin and flg22 did not induce either HR or ROS hyperaccumulation; however, INF1 accelerated HR and ROS in DS1 plants. Activation of these defense responses was closely associated with increased phosphatidic acid (PA) content. Our results show that DS1 plants exhibit PA-mediated sensitization of plant defenses and that cell death-inducing stress is required to achieve full activation of defense responses.

A receptor pair with an integrated decoy converts pathogen disabling of transcription factors to immunity.

Microbial pathogens infect host cells by delivering virulence factors (effectors) that interfere with defenses. In plants, intracellular nucleotide-binding/leucine-rich repeat receptors (NLRs) detect specific effector interference and trigger immunity by an unknown mechanism. The Arabidopsis-interacting NLR pair, RRS1-R with RPS4, confers resistance to different pathogens, including Ralstonia solanacearum bacteria expressing the acetyltransferase effector PopP2. We show that PopP2 directly acetylates a key lysine within an additional C-terminal WRKY transcription factor domain of RRS1-R that binds DNA. This disrupts RRS1-R DNA association and activates RPS4-dependent immunity. PopP2 uses the same lysine acetylation strategy to target multiple defense-promoting WRKY transcription factors, causing loss of WRKY-DNA binding and transactivating functions needed for defense gene expression and disease resistance. Thus, RRS1-R integrates an effector target with an NLR complex at the DNA to switch a potent bacterial virulence activity into defense gene activation.

Adoptive Transfer of MAGE-A4 T-cell Receptor Gene-Transduced Lymphocytes in Patients with Recurrent Esophageal Cancer.

PURPOSE: Preparative lymphodepletion, the temporal ablation of the immune system, has been reported to promote persistence of transferred cells along with increased rates of tumor regression in patients treated with adoptive T-cell therapy. However, it remains unclear whether lymphodepletion is indispensable for immunotherapy with T-cell receptor (TCR) gene-engineered T cells. EXPERIMENTAL DESIGN: We conducted a first-in-man clinical trial of TCR gene-transduced T-cell transfer in patients with recurrent MAGE-A4-expressing esophageal cancer. The patients were given sequential MAGE-A4 peptide vaccinations. The regimen included neither lymphocyte-depleting conditioning nor administration of IL2. Ten patients, divided into 3 dose cohorts, received T-cell transfer. RESULTS: TCR-transduced cells were detected in the peripheral blood for 1 month at levels proportional to the dose administered, and in 5 patients they persisted for more than 5 months. The persisting cells maintained ex vivo antigen-specific tumor reactivity. Despite the long persistence of the transferred T cells, 7 patients exhibited tumor progression within 2 months after the treatment. Three patients who had minimal tumor lesions at baseline survived for more than 27 months. CONCLUSIONS: These results suggest that TCR-engineered T cells created by relatively short-duration in vitro culture of polyclonal lymphocytes in peripheral blood retained the capacity to survive in a host. The discordance between T-cell survival and tumor regression suggests that multiple mechanisms underlie the benefits of preparative lymphodepletion in adoptive T-cell therapy.

Kinase-mediated orchestration of NADPH oxidase in plant immunity.

Reactive oxygen species (ROS) are important signalling molecules, which participate in multiple physiological processes including immune response, development, cell elongation and hormonal signalling in plants. Plant NADPH oxidase, termed respiratory burst oxidase homologue (RBOH), is frequently studied as a main player for pathogen-responsive ROS burst. Our understanding of the activation mechanism of RBOH after pathogen recognition has increased in recent years. In this review, we focus on kinase-mediated regulatory mechanisms of RBOHs. Calcium-dependent protein kinases (CDPKs) are well known to activate RBOHs by direct phosphorylation. In addition to functions of CDPKs in plants, we also describe the involvement of receptor-like cytoplasmic kinases (RLCKs) and mitogen-activated protein kinases (MAPKs) in fine-tuning RBOH activity at the post-translational and transcriptional levels, respectively.

Map-based cloning and characterization of a brown planthopper resistance gene BPH26 from Oryza sativa L. ssp. indica cultivar ADR52.

The brown planthopper (BPH) is the most serious insect pest of rice in Asia. The indica rice cultivar ADR52 carries two BPH resistance genes, BPH26 (brown planthopper resistance 26) and BPH25. Map-based cloning of BPH26 revealed that BPH26 encodes a coiled-coil-nucleotide-binding-site-leucine-rich repeat (CC-NBS-LRR) protein. BPH26 mediated sucking inhibition in the phloem sieve element. BPH26 was identical to BPH2 on the basis of DNA sequence analysis and feeding ability of the BPH2-virulent biotype of BPH. BPH2 was widely incorporated in elite rice cultivars and was well-cultivated in many Asian countries as a favorable gene resource in rice breeding against BPH. However, BPH2 was rendered ineffective by a virulent biotype of BPH in rice fields in Asia. In this study, we suggest that BPH2 can be reused by combining with other BPH resistance genes, such as BPH25, to ensure durable resistance to BPH.

In vivo phosphorylation of WRKY transcription factor by MAPK.

Plants activate signaling networks in response to diverse pathogen-derived signals, facilitating transcriptional reprogramming through mitogen-activated protein kinase (MAPK) cascades. Identification of phosphorylation targets of MAPK and in vivo detection of the phosphorylated substrates are important processes to elucidate the signaling pathway in plant immune responses. We have identified a WRKY transcription factor, which is phosphorylated by defense-related MAPKs, SIPK and WIPK. Recent evidence demonstrated that some group I WRKY transcription factors, which contain a conserved motif in the N-terminal region, are activated by MAPK-dependent phosphorylation. In this chapter, we describe protocols for preparation of anti-phosphopeptide antibodies, detection of activated MAPKs using anti-phospho-MAPK antibody, and activated WRKY using anti-phospho-WRKY antibody, respectively.

SEC14 phospholipid transfer protein is involved in lipid signaling-mediated plant immune responses in Nicotiana benthamiana.

We previously identified a gene related to the SEC14-gene phospholipid transfer protein superfamily that is induced in Nicotiana benthamiana (NbSEC14) in response to infection with Ralstonia solanacearum. We here report that NbSEC14 plays a role in plant immune responses via phospholipid-turnover. NbSEC14-silencing compromised expression of defense-related PR-4 and accumulation of jasmonic acid (JA) and its derivative JA-Ile. Transient expression of NbSEC14 induced PR-4 gene expression. Activities of diacylglycerol kinase, phospholipase C and D, and the synthesis of diacylglycerol and phosphatidic acid elicited by avirulent R. solanacearum were reduced in NbSEC14-silenced plants. Accumulation of signaling lipids and activation of diacylglycerol kinase and phospholipases were enhanced by transient expression of NbSEC14. These results suggest that the NbSEC14 protein plays a role at the interface between lipid signaling-metabolism and plant innate immune responses.

Silencing of DS2 aminoacylase-like genes confirms basal resistance to Phytophthora infestans in Nicotiana benthamiana.

Nicotiana benthamiana is a potential host to several plant pathogens, and immature leaves of N. benthamiana are susceptible to Phytophthora infestans. In contrast, mature leaves of N. benthamiana are weakly susceptible and show basal resistance to P. infestans. We screened a gene-silenced mature plant showing high resistance to P. infestans, designated as DS2 (Disease suppression 2). The deduced amino acid sequence of cDNA responsible for DS2 encoded a putative aminoacylase. Growth of P. infestans decreased in DS2 plants. Trypan blue staining revealed inhibited hyphae growth of P. infestans with an increased number of dead cells under the penetration site in DS2 plants. Consistent with growth inhibition of P. infestans, defense responses such as reactive oxygen generation and expression of a salicylic acid-dependent PR-1a increased markedly in DS2 plants compared with that of control plants. DS2 phenotype was compromised in NahG plants, suggesting DS2 phenotype depends on the salicylic acid signaling pathway. Accelerated defense response was observed in DS2 plants elicited by INF1 elicitin as well as by NbMEK2(DD), which is the constitutive active form of NbMEK2, and act as a downstream regulator of INF1 perception. On the other hand, INF1- and NbMEK2(DD)-induced defense responses were prevented by DS2-overexpressing transgenic tobacco. These results suggest that DS2 negatively regulates plant defense responses against P. infestans via NbMEK2 and SA-dependent signaling pathway in N. benthamiana.

Suppression of DS1 phosphatidic acid phosphatase confirms resistance to Ralstonia solanacearum in Nicotiana benthamiana.

Nicotianabenthamiana is susceptible to Ralstonia solanacearum. To analyze molecular mechanisms for disease susceptibility, we screened a gene-silenced plant showing resistance to R. solanacearum, designated as DS1 (Disease suppression 1). The deduced amino acid sequence of DS1 cDNA encoded a phosphatidic acid phosphatase (PAP) 2. DS1 expression was induced by infection with a virulent strain of R. solanacearum in an hrp-gene-dependent manner. DS1 rescued growth defects of the temperature-sensitive ∆lpp1∆dpp1∆pah1 mutant yeast. Recombinant DS1 protein showed Mg(2+)-independent PAP activity. DS1 plants showed reduced PAP activity and increased phosphatidic acid (PA) content. After inoculation with R. solanacearum, DS1 plants showed accelerated cell death, over-accumulation of reactive oxygen species (ROS), and hyper-induction of PR-4 expression. In contrast, DS1-overexpressing tobacco plants showed reduced PA content, greater susceptibility to R. solanacearum, and reduced ROS production and PR-4 expression. The DS1 phenotype was partially compromised in the plants in which both DS1 and NbCoi1 or DS1 and NbrbohB were silenced. These results show that DS1 PAP may affect plant immune responses related to ROS and JA cascades via regulation of PA levels. Suppression of DS1 function or DS1 expression could rapidly activate plant defenses to achieve effective resistance against Ralstonia solanacearum.

A translationally controlled tumor protein negatively regulates the hypersensitive response in Nicotiana benthamiana.

We have been isolating and characterizing Ralstonia solanacearum-responsive genes (RsRGs) in Nicotiana plants. In this study we focused on RsRG308, which we renamed NbTCTP (N. benthamiana translationally controlled tumor protein) because it encodes a polypeptide showing similarity to translationally controlled tumor proteins. Induction of the hypersensitive response (HR) was accelerated in NbTCTP-silenced N. benthamiana plants challenged with R. solanacearum 8107 (Rs8107). The Rs8107 population decreased significantly, whereas hin1 gene expression was enhanced in the silenced plant. Accelerated induction of HR was observed in NbTCTP-silenced plants inoculated with Pseudomonas cichorii and P. syringae pv. syringae. Silencing of NbTCTP also accelerated the induction of HR cell death by Agrobacterium-mediated transient expression of HR inducers, such as AvrA, BAX, INF1 and NbMEK2(DD). NbTCTP silencing enhanced NbrbohB- and NbMEK2-mediated reactive oxygen species production, leading to HR. Transient expression of both the full-length sequence and the Bcl-xL domain of NbTCTP decreased HR cell death induced by Agrobacterium-mediated transient expression of HR inducers. NbTCTP-silenced plants also showed slightly dwarf phenotypes. Therefore, NbTCTP might have a role in cell death regulation during HR to fine-tune programmed cell death-associated plant defense responses.

The variable domain of a plant calcium-dependent protein kinase (CDPK) confers subcellular localization and substrate recognition for NADPH oxidase.

Calcium-dependent protein kinases (CDPKs) are Ca(2+) sensors that regulate diverse biological processes in plants and apicomplexans. However, how CDPKs discriminate specific substrates in vivo is still largely unknown. Previously, we found that a potato StCDPK5 is dominantly localized to the plasma membrane and activates the plasma membrane NADPH oxidase (RBOH; for respiratory burst oxidase homolog) StRBOHB by direct phosphorylation of the N-terminal region. Here, we report the contribution of the StCDPK5 N-terminal variable (V) domain to activation of StRBOHB in vivo using heterologous expression system in Nicotiana benthamiana. Mutations of N-terminal myristoylation and palmitoylation sites in the V domain eliminated the predominantly plasma membrane localization and the capacity of StCDPK5 to activate StRBOHB in vivo. A tomato SlCDPK2, which also contains myristoylation and palmitoylation sites in its N terminus, phosphorylated StRBOHB in vitro but not in vivo. Functional domains responsible for activation and phosphorylation of StRBOHB were identified by swapping regions for each domain between StCDPK5 and SlCDPK2. The substitution of the V domain of StCDPK5 with that of SlCDPK2 abolished the activation and phosphorylation abilities of StRBOHB in vivo and relocalized the chimeric CDPK to the trans-Golgi network, as observed for SlCDPK2. Conversely, SlCDPK2 substituted with the V domain of StCDPK5 localized to the plasma membrane and activated StRBOHB. These results suggest that the V domains confer substrate specificity in vivo by dictating proper subcellular localization of CDPKs.