The prodomain of Arabidopsis metacaspase 2 positively regulates immune signaling mediated by pattern-recognition receptors.

Metacaspases (MCs) are structural homologs of mammalian caspases found in plants, fungi, and protozoa. Type-I MCs carry an N-terminal prodomain, the function of which is unclear. Through genetic analysis of Arabidopsis mc2-1, a T-DNA insertion mutant of MC2, we demonstrated that the prodomain of metacaspase 2 (MC2) promotes immune signaling mediated by pattern-recognition receptors (PRRs). In mc2-1, immune responses are constitutively activated. The receptor-like kinases (RLKs) BAK1/BKK1 and SOBIR1 are required for the autoimmune phenotype of mc2-1, suggesting that immune signaling mediated by the receptor-like protein (RLP)-type PRRs is activated in mc2-1. A suppressor screen identified multiple mutations in the first exon of MC2, which suppress the autoimmunity in mc2-1. Further analysis revealed that the T-DNA insertion at the end of exon 1 of MC2 causes elevated expression of the MC2 prodomain, and overexpression of the MC2 prodomain in wild-type (WT) plants results in the activation of immune responses. The MC2 prodomain interacts with BIR1, which inhibits RLP-mediated immune signaling by interacting with BAK1, suggesting that the MC2 prodomain promotes plant defense responses by interfering with the function of BIR1. Our study uncovers an unexpected function of the prodomain of a MC in plant immunity.

Antagonistic Regulation of ABA Responses by Duplicated Tandemly Repeated DUF538 Protein Genes in Arabidopsis.

The plant hormone ABA (abscisic acid) regulates plant responses to abiotic stresses by regulating the expression of ABA response genes. However, the functions of a large portion of ABA response genes have remained unclear. We report in this study the identification of ASDs (ABA-inducible signal peptide-containing DUF538 proteins), a subgroup of DUF538 proteins with a signal peptide, as the regulators of plant responses to ABA in Arabidopsis. ASDs are encoded by four closely related DUF538 genes, with ASD1/ASD2 and ASD3/ASD4 being two pairs of duplicated tandemly repeated genes. The quantitative RT-PCR (qRT-PCR) results showed that the expression levels of ASDs increased significantly in response to ABA as well as NaCl and mannitol treatments, with the exception that the expression level of ASD2 remained largely unchanged in response to NaCl treatment. The results of Arabidopsis protoplast transient transfection assays showed that ASDs were localized on the plasma membrane and in the cytosol and nucleus. When recruited to the promoter of the reporter gene via a fused GD domain, ASDs were able to slightly repress the expression of the co-transfected reporter gene. Seed germination and cotyledon greening assays showed that ABA sensitivity was increased in the transgenic plants that were over-expressing ASD1 or ASD3 but decreased in the transgenic plants that were over-expressing ASD2 or ASD4. On the other hand, ABA sensitivity was increased in the CRISPR/Cas9 gene-edited asd2 single mutants but decreased in the asd3 single mutants. A transcriptome analysis showed that differentially expressed genes in the 35S:ASD2 transgenic plant seedlings were enriched in several different processes, including in plant growth and development, the secondary metabolism, and plant hormone signaling. In summary, our results show that ASDs are ABA response genes and that ASDs are involved in the regulation of plant responses to ABA in Arabidopsis; however, ASD1/ASD3 and ASD2/ASD4 have opposite functions.

BIC2, a Cryptochrome Function Inhibitor, Is Involved in the Regulation of ABA Responses in Arabidopsis.

The plant hormone ABA (abscisic acid) is able to regulate plant responses to abiotic stresses via regulating the expression of ABA response genes. BIC1 (Blue-light Inhibitor of Cryptochromes 1) and BIC2 have been identified as the inhibitors of plant cryptochrome functions, and are involved in the regulation of plant development and metabolism in Arabidopsis . In this study, we report the identification of BIC2 as a regulator of ABA responses in Arabidopsis . RT-PCR (Reverse Transcription-Polymerase Chain Reaction) results show that the expression level of BIC1 remained largely unchanged, but that of BIC2 increased significantly in response to ABA treatment. Transfection assays in Arabidopsis protoplasts show that both BIC1 and BIC2 were mainly localized in the nucleus, and were able to activate the expression of the co-transfected reporter gene. Results in seed germination and seedling greening assays show that ABA sensitivity was increased in the transgenic plants overexpressing BIC2, but increased slightly, if any, in the transgenic plants overexpressing BIC1. ABA sensitivity was also increased in the bic2 single mutants in seedling greening assays, but no further increase was observed in the bic1 bic2 double mutants. On the other hand, in root elongation assays, ABA sensitivity was decreased in the transgenic plants overexpressing BIC2, as well as the bic2 single mutants, but no further decrease was observed in the bic1 bic2 double mutants. By using qRT-PCR (quantitative RT-PCR), we further examined how BIC2 may regulate ABA responses in Arabidopsis , and found that inhibition of ABA on the expression of the ABA receptor genes PYL4 (PYR1-Like 4) and PYL5 were decreased, but promotion of ABA on the expression of the protein kinase gene SnRK2.6 (SNF1-Related Protein Kinases 2.6) was enhanced in both the bic1 bic2 double mutants and 35S:BIC2 overexpression transgenic plants. Taken together, our results suggest that BIC2 regulates ABA responses in Arabidopsis possibly by affecting the expression of ABA signaling key regulator genes.

ASR1 and ASR2, Two Closely Related ABA-Induced Serine-Rich Transcription Repressors, Function Redundantly to Regulate ABA Responses in Arabidopsis.

The plant hormone abscisic acid (ABA) is able to regulate the expression of ABA-responsive genes via signaling transduction, and thus plays an important role in regulating plant responses to abiotic stresses. Hence, characterization of unknown ABA response genes may enable us to identify novel regulators of ABA and abiotic stress responses. By using RT-PCR analysis, we found that the expression levels of ABA-induced Serine-rich Repressor 1 (ASR1)and ASR2, two closely related unknown function genes, were increased in response to ABA treatment. Amino acid sequence analyses show that ASR1 contains an L×L×L motif and both ASR1 and ASR2 are enriched in serine. Transfection assays in Arabidopsis leaf protoplasts show that ASR1 and ASR2 were predominantly localized in the nucleus and were able to repress the expression of the reporter gene. The roles of ASRs in regulating ABA responses were examined by generating transgenic Arabidopsis plants over-expressing ASR1 and ASR2, respectively, and CRISPR/Cas9 gene-edited single and double mutants for ASR1 and ASR2. In both the seed germination and cotyledon greening assays, ABA sensitivity remained largely unchanged in the over-expression transgenic plants and the single mutants of ASR1 and ASR2, but greatly increased ABA sensitivity was observed in the asr1 asr2 double mutants. In root elongation assays, however, decreased ABA sensitivity was observed in the 35S:ASR1 and 35S:ASR2 transgenic plants, whereas increased ABA sensitivity was observed in the asr1 and asr2 single mutants, and ABA sensitivity was further increased in the asr1 asr2 double mutants. Transcriptome analysis show that the differentially expressed genes (DEGs) down-regulated in the 35S:ASR1 transgenic plant seedlings, but up-regulated in the asr1 asr2 double mutant seedlings were highly enriched in processes including responses to plant hormones and stress stimuli. Taken together, our results show that ASR1 and ASR2 are closely related ABA response genes, ASR1 and ASR2 are serine-rich novel transcription repressors, and they negatively regulate ABA responses in Arabidopsis in a redundant manner.

AtbZIP62 Acts as a Transcription Repressor to Positively Regulate ABA Responses in Arabidopsis.

The basic region/leucine zipper (bZIP) transcription factor AtbZIP62 is involved in the regulation of plant responses to abiotic stresses, including drought and salinity stresses, NO3 transport, and basal defense in Arabidopsis. It is unclear if it plays a role in regulating plant responses to abscisic acid (ABA), a phytohormone that can regulate plant abiotic stress responses via regulating downstream ABA-responsive genes. Using RT-PCR analysis, we found that the expression level of AtbZIP62 was increased in response to exogenously applied ABA. Protoplast transfection assays show that AtbZIP62 is predominantly localized in the nucleus and functions as a transcription repressor. To examine the roles of AtbZIP62 in regulating ABA responses, we generated transgenic Arabidopsis plants overexpressing AtbZIP62 and created gene-edited atbzip62 mutants using CRISPR/Cas9. We found that in both ABA-regulated seed germination and cotyledon greening assays, the 35S:AtbZIP62 transgenic plants were hypersensitive, whereas atbzip62 mutants were hyposensitive to ABA. To examine the functional mechanisms of AtbZIP62 in regulating ABA responses, we generated Arabidopsis transgenic plants overexpressing 35S:AtbZIP62-GR, and performed transcriptome analysis to identify differentially expressed genes (DEGs) in the presence and absence of DEX, and found that DEGs are highly enriched in processes including response to abiotic stresses and response to ABA. Quantitative RT-PCR results further show that AtbZIP62 may regulate the expression of several ABA-responsive genes, including USP, ABF2, and SnRK2.7. In summary, our results show that AtbZIP62 is an ABA-responsive gene, and AtbZIP62 acts as a transcription repressor to positively regulate ABA responses in Arabidopsis.

AtS40-1, a group I DUF584 protein positively regulates ABA response and salt tolerance in Arabidopsis.

Abiotic stresses such as salt and drought affect plants growth and development, whereas the plant hormone ABA is able to regulate plant responses to abiotic stresses by regulating downstream gene expression. Therefore characterization of unknown function ABA responsive genes is able to identify novel regulators of plant abiotic stress responses. We report here the characterization of AtS40-1, a Group I DUF584 protein in the regulation of ABA and salt responses in Arabidopsis. RT-PCR results show that the expression of AtS40-1 was dramatically induced by ABA, but only slightly increase, if any, was observed for other three Group I DUF584 genes including AtS40-1L, AtS40-2 and AtS40-3. Transfection assays in Arabidopsis protoplasts show that all the four Group I DUF584 proteins were predominately localized in nucleus and were able to repress the expression of the co-transfected reporter gene. The roles of AtS40-1 in regulating plant response to ABA and abiotic stress responses were analyzed, by using transgenic plants and inactivation mutants. The results show that the ABA responses were increased in the 35S:AtS40-1 transgenic plants, but decreased in the ats40-1 mutants. Similar to AtS40-1, the results indicate that AtS40-1L, the most closely related DUF584 protein to AtS40-1, positively regulates ABA responses in Arabidopsis. However, further decreased ABA responses were not observed in the ats40-1 ats40-1L double mutants. On the other hand, salt tolerance was increased in the transgenic plants overexpressing AtS40-1 or AtS40-1L, but decreased in the ats40-1 and ats40-1L mutants. Quantitative RT-PCR results show that the ABA induced expression of the ABA signaling regulator genes ABI3, ABI4 and ABA responsive gene RAB18 was decreased, where as ABA signaling gene ABI1 was increased in the ats40-1 mutants. These results suggest that AtS40-1 regulates ABA and salt responses in Arabidopsis, possibly by affecting ABA induced expression of some ABA signaling regulator genes.

The R2R3 MYB Transcription Factor MYB71 Regulates Abscisic Acid Response in Arabidopsis.

Abscisic acid (ABA) regulates plant responses to abiotic stresses via regulating the expression of downstream genes, yet the functions of many ABA responsive genes remain unknown. We report here the characterization of MYB71, a R2R3 MYB transcription factor in regulating ABA responses in Arabidopsis. RT-PCR results show that the expression level of MYB71 was increased in response to ABA treatment. Arabidopsis protoplasts transfection results show that MYB71 was specifically localized in nucleus and it activated the Gal4:GUS reporter gene when recruited to the Gal4 promoter by a fused DNA binding domain GD. Roles of MYB71 in regulating plant response to ABA were analyzed by generating Arabidopsis transgenic plants overexpression MYB71 and gene edited mutants of MYB71. The results show that ABA sensitivity was increased in the transgenic plants overexpression MYB71, but decreased in the MYB71 mutants. By using a DEX inducible system, we further identified genes are likely regulated by MYB71, and found that they are enriched in biological process to environmental stimuli including abiotic stresses, suggesting that MYB71 may regulate plant response to abiotic stresses. Taken together, our results suggest that MYB71 is an ABA responsive gene, and MYB71 functions as a transcription activator and it positively regulates ABA response in Arabidopsis.

Mutation of GmAITR Genes by CRISPR/Cas9 Genome Editing Results in Enhanced Salinity Stress Tolerance in Soybean.

Breeding of stress-tolerant plants is able to improve crop yield under stress conditions, whereas CRISPR/Cas9 genome editing has been shown to be an efficient way for molecular breeding to improve agronomic traits including stress tolerance in crops. However, genes can be targeted for genome editing to enhance crop abiotic stress tolerance remained largely unidentified. We have previously identified abscisic acid (ABA)-induced transcription repressors (AITRs) as a novel family of transcription factors that are involved in the regulation of ABA signaling, and we found that knockout of the entire family of AITR genes in Arabidopsis enhanced drought and salinity tolerance without fitness costs. Considering that AITRs are conserved in angiosperms, AITRs in crops may be targeted for genome editing to improve abiotic stress tolerance. We report here that mutation of GmAITR genes by CRISPR/Cas9 genome editing leads to enhanced salinity tolerance in soybean. By using quantitative RT-PCR analysis, we found that the expression levels of GmAITRs were increased in response to ABA and salt treatments. Transfection assays in soybean protoplasts show that GmAITRs are nucleus proteins, and have transcriptional repression activities. By using CRISPR/Cas9 to target the six GmAITRs simultaneously, we successfully generated Cas9-free gmaitr36 double and gmaitr23456 quintuple mutants. We found that ABA sensitivity in these mutants was increased. Consistent with this, ABA responses of some ABA signaling key regulator genes in the gmaitr mutants were altered. In both seed germination and seedling growth assays, the gmaitr mutants showed enhanced salt tolerance. Most importantly, enhanced salinity tolerance in the mutant plants was also observed in the field experiments. These results suggest that mutation of GmAITR genes by CRISPR/Cas9 is an efficient way to improve salinity tolerance in soybean.

Arabidopsis CALMODULIN-BINDING PROTEIN 60b plays dual roles in plant immunity.

Arabidopsis SYSTEMIC ACQUIRED RESISTANCE DEFICIENT 1 (SARD1) and CALMODULIN-BINDING PROTEIN 60g (CBP60g) are two master transcription factors that regulate many defense-related genes in plant immunity. They are required for immunity downstream of the receptor-like protein SUPPRESSOR OF NPR1-1, CONSTITUTIVE 2 (SNC2). Constitutive defense responses in the gain-of-function autoimmune snc2-1D mutant are modestly affected in either sard1 or cbp60g single mutants but completely suppressed in the sard1 cbp60g double mutant. Here we report that CBP60b, another member of the CBP60 family, also functions as a positive regulator of SNC2-mediated immunity. Loss-of-function mutations of CBP60b suppress the constitutive expression of SARD1 and enhanced disease resistance in cbp60g-1 snc2-1D, whereas overexpression of CBP60b leads to elevated SARD1 expression and constitutive defense responses. In addition, transient expression of CBP60b in Nicotiana benthamiana activates the expression of the pSARD1::luciferase reporter gene. Chromatin immunoprecipitation assays further showed that CBP60b is recruited to the promoter region of SARD1, suggesting that it directly regulates SARD1 expression. Interestingly, knocking out CBP60b in the wild-type background leads to ENHANCED DISEASE SUSCEPTIBILITY 1 (EDS1)-dependent autoimmunity, suggesting that CBP60b is required for the expression of a guardee/decoy or a negative regulator of immunity mediated by receptors carrying an N-terminal Toll-interleukin-1 receptor-like domain.

The Carboxyl-Terminus of TRANSPARENT TESTA GLABRA1 Is Critical for Its Functions in Arabidopsis.

The Arabidopsis WD40 repeat protein TRANSPARENT TESTA GLABRA1 (TTG1) regulates cell fate determination, including trichome initiation and root hair formation, as well as secondary metabolism such as flavonoid biosynthesis and seed coat mucilage production. TTG1 regulates different processes via regulating the expression of its downstream target genes by forming MYB-bHLH-WD40 (MBW) activator complexes with different R2R3 MYB and bHLH transcription factors. Here, we report the identification of the carboxyl (C)-terminus as a critical domain for TTG1's functions in Arabidopsis. We found that the ttg1Δ15aa mutant shows pleiotropic phenotypes identical to a TTG1 loss-of-function mutant. Gene sequencing indicates that a single nucleotide substitution in TTG1 led to a premature stop at the W327 residue, leading to the production of a truncated TTG1 protein with a deletion of the last 15 C-terminal amino acids. The expression of TTG1 under the control of its native promoter fully restored the ttg1Δ15aa mutant phenotypes. Consistent with these observations, the expression levels of TTG1 downstream genes such as GLABRA2 (GL2) and CAPRICE (CPC) were reduced in the ttg1Δ15aa mutant. Assays in Arabidopsis protoplast show that TTG1Δ15aa failed to interact with the bHLH transcription factor GL3, and the deletion of the last 3 C-terminal amino acids or the 339L amino acid alone fully abolished the interaction of TTG1 with GL3. Furthermore, the expression of TTG1Δ3aa under the control of TTG1 native promoter failed to restore the ttg1Δ15aa mutant phenotypes. Taken together, our results suggest that the C-terminal domain of TTG1 is required for its proper function in Arabidopsis.

Activation of TIR signalling boosts pattern-triggered immunity.

Plant immune responses are mainly activated by two types of receptor. Pattern recognition receptors localized on the plasma membrane perceive extracellular microbial features, and nucleotide-binding leucine-rich repeat receptors (NLRs) recognize intracellular effector proteins from pathogens1. NLRs possessing amino-terminal Toll/interleukin-1 receptor (TIR) domains activate defence responses via the NADase activity of the TIR domain2,3. Here we report that activation of TIR signalling has a key role in pattern-triggered immunity (PTI) mediated by pattern recognition receptors. TIR signalling mutants exhibit attenuated PTI responses and decreased resistance against pathogens. Consistently, PTI is compromised in plants with reduced NLR levels. Treatment with the PTI elicitor flg22 or nlp20 rapidly induces many genes encoding TIR-domain-containing proteins, which is likely to be responsible for activating TIR signalling during PTI. Overall, our study reveals that activation of TIR signalling is an important mechanism for boosting plant defence during PTI.

WRKY54 and WRKY70 positively regulate SARD1 and CBP60g expression in plant immunity.

SARD1 and CBP60g are two master regulators in plant immunity. They are required for the constitutive defense responses in the Arabidopsis snc2-1D mutant, which carries a gain-of-function mutation in a receptor-like protein. Here we report that WRKY54 and WRKY70 are required for activation of SARD1 and CBP60g expression and defense responses in snc2-1D. In addition, the induction of SARD1 and CBP60g by the bacterial pathogen Pseudomonas syringae pv. maculicola is significantly reduced in sid2 wrky54 wrky70 triple mutants compared to the sid2 single mutants, suggesting that WRKY54 and WRKY70 positively regulate the SID2-independent expression of SARD1 and CBP60g during pathogen infection. Our study revealed WRKY54 and WRKY70 as positive regulators of SARD1 and CBP60g expression in plant defense.

PIP2, An Auxin Induced Plant Peptide Hormone Regulates Root and Hypocotyl Elongation in Arabidopsis.

Auxin is one of the traditional plant hormones, whereas peptide hormones are peptides with hormone activities. Both auxin and plant peptide hormones regulate multiple aspects of plant growth and development, and there are cross-talks between auxin and plant peptide hormones. PAMP-INDUCED SECRETED PEPTIDES (PIPs) and PIP-LIKEs (PIPLs) are a new family of plant peptide hormone, and PIPL3/TARGET OF LBD SIXTEEN 2 (TOLS2) has been shown to regulate lateral root formation in Arabidopsis. We report here the identification of PIP2 as an auxin response gene, and we found it plays a role in regulating root and hypocotyl development in Arabidopsis. By using quantitative RT-PCR, we found that the expression of PIP2 but not PIP1 and PIP3 was induced by auxin, and auxin induced expression of PIP2 was reduced in nph4-1 and arf19-4, the lost-of-function mutants of Auxin Response Factor 7 (ARF7) and ARF19, respectively. By generating and characterizing overexpressing transgenic lines and gene edited mutants for PIP2, we found that root length in the PIP2 overexpression plant seedlings was slightly shorter when compared with that in the Col wild type plants, but root length of the pip2 mutant seedlings remained largely unchanged. For comparison, we also generated overexpressing transgenic lines and gene edited mutants for PIP3, as well as pip2 pip3 double mutants. Surprisingly, we found that root length in the PIP3 overexpression plant seedlings is shorter than that of the PIP2 overexpression plant seedlings, and the pip3 mutant seedlings also produced short roots. However, root length in the pip2 pip3 double mutant seedlings is largely similar to that in the pip3 single mutant seedlings. On the other hand, hypocotyl elongation assays indicate that only the 35S:PIP2 transgenic plant seedlings produced longer hypocotyls when compared with the Col wild type seedlings. Further analysis indicates that PIP2 promotes cell division as well as cell elongation in hypocotyls. Taken together, our results suggest that PIP2 is an auxin response gene, and PIP2 plays a role in regulating root and hypocotyl elongation in Arabidopsis likely via regulating cell division and cell elongation.

The glycosyltransferase UGT76B1 modulates N-hydroxy-pipecolic acid homeostasis and plant immunity.

The tradeoff between growth and defense is a critical aspect of plant immunity. Therefore, the plant immune response needs to be tightly regulated. Salicylic acid (SA) is an important plant hormone regulating defense against biotrophic pathogens. Recently, N-hydroxy-pipecolic acid (NHP) was identified as another regulator for plant innate immunity and systemic acquired resistance (SAR). Although the biosynthetic pathway leading to NHP formation is already been identified, how NHP is further metabolized is unclear. Here, we present UGT76B1 as a uridine diphosphate-dependent glycosyltransferase (UGT) that modifies NHP by catalyzing the formation of 1-O-glucosyl-pipecolic acid in Arabidopsis thaliana. Analysis of T-DNA and clustered regularly interspaced short palindromic repeats (CRISPR) knock-out mutant lines of UGT76B1 by targeted and nontargeted ultra-high performance liquid chromatography coupled to high-resolution mass spectrometry (UHPLC-HRMS) underlined NHP and SA as endogenous substrates of this enzyme in response to Pseudomonas infection and UV treatment. ugt76b1 mutant plants have a dwarf phenotype and constitutive defense response which can be suppressed by loss of function of the NHP biosynthetic enzyme FLAVIN-DEPENDENT MONOOXYGENASE 1 (FMO1). This suggests that elevated accumulation of NHP contributes to the enhanced disease resistance in ugt76b1. Externally applied NHP can move to distal tissue in ugt76b1 mutant plants. Although glycosylation is not required for the long-distance movement of NHP during SAR, it is crucial to balance growth and defense.

Knockout of the entire family of AITR genes in Arabidopsis leads to enhanced drought and salinity tolerance without fitness costs.

BACKGORUND: Environmental stresses including abiotic stresses and biotic stresses limit yield of plants. Stress-tolerant breeding is an efficient way to improve plant yield under stress conditions. Genome editing by CRISPR/Cas9 can be used in molecular breeding to improve agronomic traits in crops, but in most cases, with fitness costs. The plant hormone ABA regulates plant responses to abiotic stresses via signaling transduction. We previously identified AITRs as a family of novel transcription factors that play a role in regulating plant responses to ABA and abiotic stresses. We found that abiotic stress tolerance was increased in the single, double and triple aitr mutants. However, it is unclear if the increased abiotic stress tolerance in the mutants may have fitness costs. RESULTS: We report here the characterization of AITRs as suitable candidate genes for CRISPR/Cas9 editing to improve plant stress tolerance. By using CRISPR/Cas9 to target AITR3 and AITR4 simultaneously in the aitr256 triple and aitr1256 quadruple mutants respectively, we generated Cas9-free aitr23456 quintuple and aitr123456 sextuple mutants. We found that reduced sensitivities to ABA and enhanced tolerance to drought and salt were observed in these mutants. Most importantly, plant growth and development was not affected even in the aitr123456 sextuple mutants, in whom the entire AITR family genes have been knocked out, and the aitr123456 sextuple mutants also showed a wild type response to the pathogen infection. CONCLUSIONS: Our results suggest that knockout of the AITR family genes in Arabidopsis enhanced abiotic stress tolerance without fitness costs. Considering that knock-out a few AITRs will lead to enhanced abiotic stress tolerance, that AITRs are widely distributed in angiosperms with multiple encoding genes, AITRs may be targeted for molecular breeding to improve abiotic stress tolerance in plants including crops.

AITRL, an evolutionarily conserved plant specific transcription repressor regulates ABA response in Arabidopsis.

Expression of stress response genes can be regulated by abscisic acid (ABA) dependent and ABA independent pathways. Osmotic stresses promote ABA accumulation, therefore inducing the expression of stress response genes via ABA signaling. Whereas cold and heat stresses induce the expression of stress response genes via ABA independent pathway. ABA induced transcription repressors (AITRs) are a family of novel transcription factors that play a role in ABA signaling, and Drought response gene (DRG) has previously been shown to play a role in regulating plant response to drought and freezing stresses. We report here the identification of DRG as a novel transcription factor and a regulator of ABA response in Arabidopsis. We found that the expression of DRG was induced by ABA treatment. Homologs searching identified AITR5 as the most closely related Arabidopsis protein to DRG, and homologs of DRG, including the AITR-like (AITRL) proteins in bryophytes and gymnosperms, are specifically presented in embryophytes. Therefore we renamed DRG as AITRL. Protoplast transfection assays show that AITRL functioned as a transcription repressor. In seed germination and seedling greening assays, the aitrl mutants showed an increased sensitivity to ABA. By using qRT-PCR, we show that ABA responses of some ABA signaling component genes including some PYR1-likes (PYLs), PROTEIN PHOSPHATASE 2Cs (PP2Cs) and SUCROSE NONFERMENTING 1 (SNF1)-RELATED PROTEIN KINASES 2s (SnRK2s) were reduced in the aitrl mutants. Taken together, our results suggest that AITRLs are a family of novel transcription repressors evolutionally conserved in embryophytes, and AITRL regulates ABA response in Arabidopsis by affecting ABA response of some ABA signaling component genes.

Receptor-like kinases MDS1 and MDS2 promote SUMM2-mediated immunity.

Disruption of the MEKK1-MKK1/MKK2-MPK4 kinase cascade leads to activation of immunity mediated by the nucleotide-binding leucine-rich repeat (NLR) immune receptor SUMM2, which monitors the phosphorylation status of CRCK3. Here we report that two receptor-like kinases (RLKs), MDS1, and MDS2, function redundantly to promote SUMM2-mediated immunity. Activation of SUMM2-mediated immunity is dependent on MDS1, and to a less extent on MDS2. MDS1 associates with CRCK3 in planta and can phosphorylate CRCK3 in vitro, suggesting that it may target CRCK3 to positively regulate SUMM2-mediated signaling. Our finding highlights a new defense mechanism where RLKs promote NLR-mediated immunity.

Diverse Roles of the Salicylic Acid Receptors NPR1 and NPR3/NPR4 in Plant Immunity.

The plant defense hormone salicylic acid (SA) is perceived by two classes of receptors, NPR1 and NPR3/NPR4. They function in two parallel pathways to regulate SA-induced defense gene expression. To better understand the roles of the SA receptors in plant defense, we systematically analyzed their contributions to different aspects of Arabidopsis (Arabidopsis thaliana) plant immunity using the SA-insensitive npr1-1 npr4-4D double mutant. We found that perception of SA by NPR1 and NPR4 is required for activation of N-hydroxypipecolic acid biosynthesis, which is essential for inducing systemic acquired resistance. In addition, both pattern-triggered immunity (PTI) and effector-triggered immunity (ETI) are severely compromised in the npr1-1 npr4-4D double mutant. Interestingly, the PTI and ETI attenuation in npr1-1 npr4-4D is more dramatic compared with the SA-induction deficient2-1 (sid2-1) mutant, suggesting that the perception of residual levels of SA in sid2-1 also contributes to immunity. Furthermore, NPR1 and NPR4 are involved in positive feedback amplification of SA biosynthesis and regulation of SA homeostasis through modifications including 5-hydroxylation and glycosylation. Thus, the SA receptors NPR1 and NPR4 play broad roles in plant immunity.

TRANSPARENT TESTA GLABRA1, a Key Regulator in Plants with Multiple Roles and Multiple Function Mechanisms.

TRANSPARENT TESTA GLABRA1 (TTG1) is a WD40 repeat protein. The phenotypes caused by loss-of-function of TTG1 were observed about half a century ago, but the TTG1 gene was identified only about twenty years ago. Since then, TTG1 has been found to be a plant-specific regulator with multiple roles and multiple functional mechanisms. TTG1 is involved in the regulation of cell fate determination, secondary metabolisms, accumulation of seed storage reserves, plant responses to biotic and abiotic stresses, and flowering time in plants. In some processes, TTG1 may directly or indirectly regulate the expression of downstream target genes via forming transcription activator complexes with R2R3 MYB and bHLH transcription factors. Whereas in other processes, TTG1 may function alone or interact with other proteins to regulate downstream target genes. On the other hand, the studies on the regulation of TTG1 are very limited. So far, only the B3-domain family transcription factor FUSCA3 (FUS3) has been found to regulate the expression of TTG1, phosphorylation of TTG1 affects its interaction with bHLH transcription factor TT2, and TTG1 proteins can be targeted for degradation by the 26S proteasome. Here, we provide an overview of TTG1, including the identification of TTG1, the functions of TTG1, the possible function mechanisms of TTG1, and the regulation of TTG1. We also proposed potential research directions that may shed new light on the regulation and functional mechanisms of TTG1 in plants.

Cell wall/vacuolar inhibitor of fructosidase 1 regulates ABA response and salt tolerance in Arabidopsis.

ABA regulates abiotic stress tolerance in plants via activating/repressing gene expression. However, the functions of many ABA response genes remained unknown. C/VIFs are proteinaceous inhibitors of the CWI and VI invertases. We report here the involvement of C/VIF1 in regulating ABA response and salt tolerance in Arabidopsis. We found that the expression level of C/VIF1 was increased in response to ABA treatment. By using CRISPR/Cas9 gene editing, we generated transgene-free c/vif1 mutants. We also generated C/VIF1 overexpression plants by expressing C/VIF1 under the control of the 35S promoter. We examined ABA response of the 35S:C/VIF1 transgenic plants and the c/vif1 mutants by using seed germination and seedling greening assays, and found that the 35S:C/VIF1 transgenic plants showed an enhanced sensitivity to ABA treatment in both assays. On the other hand, the c/vif1 mutants showed slight enhanced tolerance to ABA only at the early stage of germination. We also found that salt tolerance was reduced in the 35S:C/VIF1 transgenic plants in seed germination assays, but slightly increased in the c/vif1 mutants. Taken together, our results suggest that C/VIF1 is an ABA response gene, and C/VIF1 is involved in the regulation of ABA response and salt tolerance in Arabidopsis.