Quantitative Peptidomics Study Reveals That a Wound-Induced Peptide from PR-1 Regulates Immune Signaling in Tomato

Chen, Ying-Lan,Lee, Chi-Ying,Cheng, Kai-Tan,Chang, Wei-Hung,Huang, Rong-Nan,Nam, Hong Gil,Chen, Yet-Ran American Society of Plant Biologists 2014 The Plant cell Vol.26 No.10

<P>CAPE1, a conserved peptide elicitor derived from tomato PR-1, was induced by wounding and found to regulate immune responses against biological threats. As PR-1 is highly conserved across many organisms and the putative peptide from AtPR1 was also found to be bioactive in <I>Arabidopsis</I>, the results suggest that this peptide may be useful for enhancing resistance to stress in other plant species.</P><P>Many important cell-to-cell communication events in multicellular organisms are mediated by peptides, but only a few peptides have been identified in plants. In an attempt to address the difficulties in identifying plant signaling peptides, we developed a novel peptidomics approach and used this approach to discover defense signaling peptides in plants. In addition to the canonical peptide systemin, several novel peptides were confidently identified in tomato (<I>Solanum lycopersicum</I>) and quantified to be induced by both wounding and methyl jasmonate (MeJA). A wounding or wounding plus MeJA-induced peptide derived from the pathogenesis-related protein 1 (PR-1) family was found to induce significant antipathogen and minor antiherbivore responses in tomato. This study highlights a role for PR-1 in immune signaling and suggests the potential application of plant endogenous peptides in efforts to defeat biological threats in crop production. As PR-1 is highly conserved across many organisms and the putative peptide from At-PR1 was also found to be bioactive in <I>Arabidopsis thaliana</I>, our results suggest that this peptide may be useful for enhancing resistance to stress in other plant species.</P>

Inverse Correlation Between MPSR1 E3 Ubiquitin Ligase and HSP90.1 Balances Cytoplasmic Protein Quality Control

Kim, Jong Hum,Oh, Tae Rin,Cho, Seok Keun,Yang, Seong Wook,Kim, Woo Taek American Society of Plant Biologists 2019 Plant Physiology Vol.180 No.2

<P>The inverse correlation between MPSR1 and AtHSP90.1 via miR414 may adjust the set-point of the HSP90-mediated protein quality control process in response to increasing stress intensity in Arabidopsis.</P><P>MISFOLDED PROTEIN SENSING RING1 (MPSR1) is a chaperone-independent E3 ubiquitin ligase that participates in protein quality control by eliminating misfolded proteins in Arabidopsis (<I>Arabidopsis thaliana</I>). Here, we report that in the early stages of proteotoxic stress, cellular levels of MPSR1 increased immediately, whereas levels of HEAT SHOCK PROTEIN90.1 (AtHSP90.1) were unaltered despite massively upregulated transcription. At this stage, the gene-silencing pathway mediated by microRNA 414 (miR414) suppressed AtHSP90.1 translation. By contrast, under prolonged stress, AtHSP90.1 was not suppressed, and instead competed with MPSR1 to act on misfolded proteins, promoting the destruction of MPSR1. Deficiency or excess of MPSR1 significantly abolished or intensified the suppression of AtHSP90.1, respectively. Similar to the <I>MPSR1</I>-overexpressing transgenic plants, the <I>miR414</I>-overexpressing plants showed an increased tolerance to proteotoxic stress as compared to the wild-type plants. Although the functional relationship between MPSR1 and miR414 remains unclear, both MPSR1 and miR414 demonstrated negative modulation of the expression of AtHSP90.1. The inverse correlation between MPSR1 and AtHSP90.1 via miR414 may adjust the set-point of the HSP90-mediated protein quality control process in response to increasing stress intensity in Arabidopsis.</P>

Perturbation of Maize Phenylpropanoid Metabolism by an AvrE Family Type III Effector from <i>Pantoea stewartii</i>

Asselin, Jo Ann E.,Lin, Jinshan,Perez-Quintero, Alvaro L.,Gentzel, Irene,Majerczak, Doris,Opiyo, Stephen O.,Zhao, Wanying,Paek, Seung-Mann,Kim, Min Gab,Coplin, David L.,Blakeslee, Joshua J.,Mackey, Da American Society of Plant Biologists 2015 Plant Physiology Vol.167 No.3

<P><I>The virulence activity of an effector protein belonging to the widely conserved AvrE family is linked to its ability to cause system-wide reprogramming of phenylpropanoid metabolism in susceptible maize seedlings.</I></P><P>AvrE family type III effector proteins share the ability to suppress host defenses, induce disease-associated cell death, and promote bacterial growth. However, despite widespread contributions to numerous bacterial diseases in agriculturally important plants, the mode of action of these effectors remains largely unknown. WtsE is an AvrE family member required for the ability of <I>Pantoea stewartii</I> ssp. <I>stewartii</I> (<I>Pnss</I>) to proliferate efficiently and cause wilt and leaf blight symptoms in maize (<I>Zea mays</I>) plants. Notably, when WtsE is delivered by a heterologous system into the leaf cells of susceptible maize seedlings, it alone produces water-soaked disease symptoms reminiscent of those produced by <I>Pnss</I>. Thus, WtsE is a pathogenicity and virulence factor in maize, and an <I>Escherichia coli</I> heterologous delivery system can be used to study the activity of WtsE in isolation from other factors produced by <I>Pnss</I>. Transcriptional profiling of maize revealed the effects of WtsE, including induction of genes involved in secondary metabolism and suppression of genes involved in photosynthesis. Targeted metabolite quantification revealed that WtsE perturbs maize metabolism, including the induction of coumaroyl tyramine. The ability of mutant WtsE derivatives to elicit transcriptional and metabolic changes in susceptible maize seedlings correlated with their ability to promote disease. Furthermore, chemical inhibitors that block metabolic flux into the phenylpropanoid pathways targeted by WtsE also disrupted the pathogenicity and virulence activity of WtsE. While numerous metabolites produced downstream of the shikimate pathway are known to promote plant defense, our results indicate that misregulated induction of phenylpropanoid metabolism also can be used to promote pathogen virulence.</P>

pTAC10, a Key Subunit of Plastid-Encoded RNA Polymerase, Promotes Chloroplast Development

Chang, Sun Hyun,Lee, Sangyool,Um, Tae Young,Kim, Ju-Kon,Do Choi, Yang,Jang, Geupil American Society of Plant Biologists 2017 PLANT PHYSIOLOGY - Vol.174 No.1

<P>Regulation of photosynthetic gene expression by plastid-encoded RNA polymerase (PEP) is essential for chloroplast development. The activity of PEP largely relies on at least 12 PEP-associated proteins (PAPs) encoded in the nuclear genome of plant cells. A recent model proposed that these PAPs regulate the establishment of the PEP complex through broad PAP-PEP or PAP-PAP interactions. In this study, we identified the Arabidopsis (Arabidopsis thaliana) seedling-lethal mutant ptac10-1, which has defects in chloroplast development, and found that the mutant phenotype is caused by the suppression of PLASTID S1 RNA-BINDING DOMAIN PROTEIN (pTAC10/PAP3). Analysis of the heterozygous mutant and pTAC10-overexpressing transgenic plants indicated that the expression level of pTAC10 is tightly linked to chloroplast development. Characterization of the interaction of pTAC10 with PAPs revealed that pTAC10 interacts with other PAPs, such as FSD2, FSD3, TrxZ, pTAC7, and pTAC14, but it does not interact with PEP core enzymes, such as rpoA and rpoB. Analysis of pTAC10 interactions using truncated pTAC10 proteins showed that the pTAC10 carboxyl-terminal region downstream of the S1 domain is involved in the pTAC10-PAP interaction. Furthermore, overexpression of truncated pTAC10s lacking the C-terminal regions downstream of the S1 domain could not rescue the ptac10-1 mutant phenotype and induced an abnormal whitening phenotype in Columbia-0 plants. Our observations suggested that these pTAC10-PAP interactions are essential for the formation of the PEP complex and chloroplast development.</P>

The Arabidopsis Transcription Factor NAC016 Promotes Drought Stress Responses by Repressing <i>AREB1</i> Transcription through a Trifurcate Feed-Forward Regulatory Loop Involving NAP

Sakuraba, Yasuhito,Kim, Ye-Sol,Han, Su-Hyun,Lee, Byoung-Doo,Paek, Nam-Chon American Society of Plant Biologists 2015 The Plant cell Vol.27 No.6

<P>The Arabidopsis transcription factor NAC016 activates drought stress responses by inducing <I>NAP</I> transcription and repressing <I>AREB1</I> transcription by binding to different regions of the <I>AREB1</I> promoter.</P><P>Drought and other abiotic stresses negatively affect plant growth and development and thus reduce productivity. The plant-specific NAM/ATAF1/2/CUC2 (NAC) transcription factors have important roles in abiotic stress-responsive signaling. Here, we show that <I>Arabidopsis thaliana</I> NAC016 is involved in drought stress responses; <I>nac016</I> mutants have high drought tolerance, and <I>NAC016</I>-overexpressing (<I>NAC016</I>-OX) plants have low drought tolerance. Using genome-wide gene expression microarray analysis and MEME motif searches, we identified the NAC016-specific binding motif (NAC16BM), GATTGGAT[AT]CA, in the promoters of genes downregulated in <I>nac016-1</I> mutants. The NAC16BM sequence does not contain the core NAC binding motif CACG (or its reverse complement CGTG). NAC016 directly binds to the NAC16BM in the promoter of <I>ABSCISIC ACID-RESPONSIVE ELEMENT BINDING PROTEIN1</I> (<I>AREB1</I>), which encodes a central transcription factor in the stress-responsive abscisic acid signaling pathway and represses <I>AREB1</I> transcription. We found that knockout mutants of the NAC016 target gene <I>NAC-LIKE, ACTIVATED BY AP3/PI</I> (<I>NAP</I>) also exhibited strong drought tolerance; moreover, NAP binds to the <I>AREB1</I> promoter and suppresses <I>AREB1</I> transcription. Taking these results together, we propose that a trifurcate feed-forward pathway involving <I>NAC016</I>, <I>NAP</I>, and <I>AREB1</I> functions in the drought stress response, in addition to affecting leaf senescence in Arabidopsis.</P>

Functional Conservation in the SIAMESE-RELATED Family of Cyclin-Dependent Kinase Inhibitors in Land Plants

Kumar, Narender,Harashima, Hirofumi,Kalve, Shweta,Bramsiepe, Jonathan,Wang, Kai,Sizani, Bulelani L.,Bertrand, Laura L.,Johnson, Matthew C.,Faulk, Christopher,Dale, Renee,Simmons, L. Alice,Churchman, M American Society of Plant Biologists 2015 The Plant cell Vol.27 No.11

<P>Although they play roles in many different developmental processes, the biochemical function of SMR-type CDK inhibitors is conserved among land plants.</P><P>The best-characterized members of the plant-specific SIAMESE-RELATED (SMR) family of cyclin-dependent kinase inhibitors regulate the transition from the mitotic cell cycle to endoreplication, also known as endoreduplication, an altered version of the cell cycle in which DNA is replicated without cell division. Some other family members are implicated in cell cycle responses to biotic and abiotic stresses. However, the functions of most SMRs remain unknown, and the specific cyclin-dependent kinase complexes inhibited by SMRs are unclear. Here, we demonstrate that a diverse group of SMRs, including an SMR from the bryophyte <I>Physcomitrella patens</I>, can complement an <I>Arabidopsis thaliana siamese</I> (<I>sim</I>) mutant and that both Arabidopsis SIM and <I>P. patens</I> SMR can inhibit CDK activity in vitro. Furthermore, we show that Arabidopsis SIM can bind to and inhibit both CDKA;1 and CDKB1;1. Finally, we show that <I>SMR2</I> acts to restrict cell proliferation during leaf growth in Arabidopsis and that <I>SIM</I>, <I>SMR1/LGO</I>, and <I>SMR2</I> play overlapping roles in controlling the transition from cell division to endoreplication during leaf development. These results indicate that differences in SMR function in plant growth and development are primarily due to differences in transcriptional and posttranscriptional regulation, rather than to differences in fundamental biochemical function.</P>

The Pepper RING-Type E3 Ligase CaAIRF1 Regulates ABA and Drought Signaling via CaADIP1 Protein Phosphatase Degradation

Lim, Chae Woo,Baek, Woonhee,Lee, Sung Chul American Society of Plant Biologists 2017 Plant Physiology Vol.173 No.4

<P>Ubiquitin-mediated protein modification occurs at multiple steps of abscisic acid (ABA) signaling. Here, we sought proteins responsible for degradation of the pepper (Capsicum annuum) type 2C protein phosphatase CaADIP1 via the 26S proteasome system. We showed that the RING-type E3 ligase CaAIRF1 (Capsicum annuum ADIP1 Interacting RING Finger Protein 1) interacts with and ubiquitinates CaADIP1. CaADIP1 degradation was slower in crude proteins from CaAIRF1-silenced peppers than in those from control plants. CaAIRF1-silenced pepper plants displayed reduced ABA sensitivity and decreased drought tolerance characterized by delayed stomatal closure and suppressed induction of ABA- and drought-responsive marker genes. In contrast, CaAIRF1-overexpressing Arabidopsis (Arabidopsis thaliana) plants exhibited ABA- hypersensitive and drought-tolerant phenotypes. Moreover, in these plants, CaADIP1-induced ABA hyposensitivity was strongly suppressed by CaAIRF1 overexpression. Our findings highlight a potential new route for fine-tune regulation of ABA signaling in pepper via CaAIRF1 and CaADIP1.</P>

STABILIZED1 Modulates Pre-mRNA Splicing for Thermotolerance

Kim, Geun-Don,Cho, Young-Hee,Lee, Byeong-Ha,Yoo, Sang-Dong American Society of Plant Biologists 2017 Plant Physiology Vol.173 No.4

<P>High-temperature stress often leads to differential RNA splicing, thus accumulating different types and/or amounts of mature mRNAs in eukaryotic cells. However, regulatory mechanisms underlying plant precursor mRNA (pre-mRNA) splicing in the environmental stress conditions remain elusive. Herein, we describe that a U5-snRNP-interacting protein homolog STABILIZED1 (STA1) has pre-mRNA splicing activity for heat-inducible transcripts including HEAT STRESS TRANSCRIPTION FACTORs and various HEAT SHOCK PROTEINs for the establishment of heat stress tolerance in Arabidopsis (Arabidopsis thaliana). Our cell-based splicing reporter assay demonstrated STA1 acts on pre-mRNA splicing for specific subsets of stress-related genes. Cellular reconstitution of heat-inducible transcription cascades supported the view that STA1-dependent premRNA splicing plays a role in DREB2A-dependent HSFA3 expression for heat-responsive gene expression. Further genetic analysis with a loss-of-function mutant sta1-1, STA1-expressing transgenic plants in Col background, and STA1-expressing transgenic plants in the sta1-1 background verified that STA1 is essential in expression of necessary genes including HSFA3 for two-step heat stress tolerance in plants. However, constitutive overexpression of the cDNA version of HSFA3 in the sta1-1 background is unable to execute plant heat stress tolerance in sta1-1. Consistently our global target analysis of STA1 showed that its splicing activity modulates a rather broad range of gene expression in response to heat treatment. The findings of this study reveal that heat-inducible STA1 activity for pre-mRNA splicing serves as a molecular regulatory mechanism underlying the plant stress tolerance to high-temperature stress.</P>

Spatiotemporal Monitoring of <i>Pseudomonas syringae</i> Effectors via Type III Secretion Using Split Fluorescent Protein Fragments

Park, Eunsook,Lee, Hye-Young,Woo, Jongchan,Choi, Doil,Dinesh-Kumar, Savithramma P. American Society of Plant Biologists 2017 The Plant cell Vol.29 No.7

<P>Pathogenic gram-negative bacteria cause serious diseases in animals and plants. These bacterial pathogens use the type III secretion system (T3SS) to deliver effector proteins into host cells; these effectors then localize to different subcellular compartments to attenuate immune responses by altering biological processes of the host cells. The fluorescent protein (FP)-based approach to monitor effectors secreted from bacteria into the host cells is not possible because the folded FP prevents effector delivery through the T3SS. Therefore, we optimized an improved variant of self-assembling split super-folder green fluorescent protein (sfGFP(OPT)) system to investigate the spatiotemporal dynamics of effectors delivered through bacterial T3SS into plant cells. In this system, effectors are fused to 11th beta-strand of super-folder GFP (sfGFP11), and when delivered into plant cells expressing sfGFP1-10 beta-strand (sfGFP1-10(OPT)), the two proteins reconstitute GFP fluorescence. We generated a number of Arabidopsis thaliana transgenic lines expressing sfGFP1-10(OPT) targeted to various subcellular compartments to facilitate localization of sfGFP11-tagged effectors delivered from bacteria. We demonstrate the efficacy of this system using Pseudomonas syringae effectors AvrB and AvrRps4 in Nicotiana benthamiana and transgenic Arabidopsis plants. The versatile split sfGFP(OPT) system described here will facilitate a better understanding of bacterial invasion strategies used to evade plant immune responses.</P>

Pepper Heat Shock Protein 70a Interacts with the Type III Effector AvrBsT and Triggers Plant Cell Death and Immunity

Kim, Nak Hyun,Hwang, Byung Kook American Society of Plant Biologists 2015 Plant Physiology Vol.167 No.2

<P><I>A pepper heat shock protein acts as a positive regulator of plant cell death and immunity signaling in response to heat stress and microbial pathogens.</I></P><P>Heat shock proteins (HSPs) function as molecular chaperones and are essential for the maintenance and/or restoration of protein homeostasis. The genus <I>Xanthomonas</I> type III effector protein AvrBsT induces hypersensitive cell death in pepper (<I>Capsicum annuum</I>). Here, we report the identification of the pepper CaHSP70a as an AvrBsT-interacting protein. Bimolecular fluorescence complementation and coimmunoprecipitation assays confirm the specific interaction between CaHSP70a and AvrBsT in planta. The CaHSP70a peptide-binding domain is essential for its interaction with AvrBsT. Heat stress (37°C) and <I>Xanthomonas campestris</I> pv <I>vesicatoria</I> (<I>Xcv</I>) infection distinctly induce <I>CaHSP70a</I> in pepper leaves. Cytoplasmic CaHSP70a proteins significantly accumulate in pepper leaves to induce the hypersensitive cell death response by <I>Xcv</I> (<I>avrBsT</I>) infection. Transient <I>CaHSP70a</I> overexpression induces hypersensitive cell death under heat stress, which is accompanied by strong induction of defense- and cell death-related genes. The <I>CaHSP70a</I> peptide-binding domain and ATPase-binding domain are required to trigger cell death under heat stress. Transient coexpression of <I>CaHSP70a</I> and <I>avrBsT</I> leads to cytoplasmic localization of the CaHSP70a-AvrBsT complex and significantly enhances <I>avrBsT</I>-triggered cell death in <I>Nicotiana benthamiana</I>. <I>CaHSP70a</I> silencing in pepper enhances <I>Xcv</I> growth but disrupts the reactive oxygen species burst and cell death response during <I>Xcv</I> infection. Expression of some defense marker genes is significantly reduced in <I>CaHSP70a</I>-silenced leaves, with lower levels of the defense hormones salicylic acid and jasmonic acid. Together, these results suggest that CaHSP70a interacts with the type III effector AvrBsT and is required for cell death and immunity in plants.</P>