Salicylic Acid Induction Research Articles

Next-generation mapping of the salicylic acid signaling hub and transcriptional cascade

For over 60 years, salicylic acid (SA) has been known as a plant immune signal required for basal and systemic acquired resistance. SA activates these immune responses by reprogramming ∼20% of the transcriptome through NPR1. However, components in the NPR1 signaling hub, which appears as nuclear condensates, and the NPR1 signaling cascade have remained elusive due to difficulties in studying this transcriptional cofactor, whose chromatin association is indirect and likely transient. To overcome this challenge, we applied TurboID to divulge the NPR1 proxiome, which detected almost all known NPR1 interactors as well as new components of transcription-related complexes. Testing of new components showed that chromatin remodeling and histone demethylation contribute to SA-induced resistance. Globally, the NPR1 proxiome has a striking similarity to the proxiome of GBPL3 that is involved in SA synthesis, except for associated transcription factors (TFs), suggesting that common regulatory modules are recruited to reprogram specific transcriptomes by transcriptional cofactors, like NPR1, through binding to unique TFs. Stepwise green fluorescent protein-tagged factor cleavage under target and release using nuclease (greenCUT&RUN) analyses showed that, upon SA induction, NPR1 initiates the transcriptional cascade primarily through association with TGACG-binding TFs to induce expression of secondary TFs, predominantly WRKYs. Further, WRKY54 and WRKY70 were identified to play a major role in inducing immune-output genes without interacting with NPR1 at the chromatin. Moreover, loss of condensate formation function of NPR1 decreases its chromatin association and transcriptional activity, indicating the importance of condensates in organizing the NPR1 signaling hub and initiating the transcriptional cascade. Collectively, this study demonstrates how combinatorial applications of TurboID and stepwise greenCUT&RUN transcend traditional genetic methods to globally map signaling hubs and transcriptional cascades for in-depth explorations.

CBP60b clade proteins are prototypical transcription factors mediating immunity.

Transcriptional reprogramming is critical for plant immunity. Several calmodulin (CaM)-binding protein 60 (CBP60) family transcription factors (TFs) in Arabidopsis (Arabidopsis thaliana), including CBP60g, Systemic Acquired Resistance Deficient 1 (SARD1), CBP60a, and CBP60b, are critical for and show distinct roles in immunity. However, there are additional CBP60 members whose function is unclear. We report here that Arabidopsis CBP60c-f, four uncharacterized CBP60 members, play redundant roles with CBP60b in the transcriptional regulation of immunity responses, whose pCBP60b-driven expression compensates the loss of CBP60b. By contrast, neither CBP60g nor SARD1 is inter-changeable with CBP60b, suggesting clade-specific functionalization. We further show that function of CBP60b clade TFs relies on DNA-binding domains (DBDs) and CaM-binding domains, suggesting that they are downstream components of calcium signaling. Importantly, we demonstrate that CBP60s encoded in earliest land plant lineage Physcomitrium patens and Selaginella moellendorffii, are functionally homologous to Arabidopsis CBP60b, suggesting that the CBP60b clade contains the prototype TFs of the CBP60 family. Furthermore, tomato and cucumber CBP60b-like genes rescue the defects of Arabidopsis cbp60b and activate the expression of tomato and cucumber SALICYLIC ACID INDUCTION DEFICIIENT2 (SID2) and ENHANCED DISEASE SUSCEPTIBILITY 1 (EDS1) genes, suggesting that immune response pathways centered on CBP60b are also evolutionarily conserved. Together, these findings suggest CBP60b clade transcription factors are functionally conserved in evolution and positively mediate immunity.

Effect of Salicylic Acid Treatment on Disease Resistance to Coleosporium zanthoxyli in Chinese Pepper (Zanthoxylum armatum).

The fungus Coleosporium zanthoxyli causes leaf rust in Chinese pepper (Zanthoxylum armatum). To investigate the control effect of elicitor treatment on leaf rust in this species, the impact of salicylic acid (SA) on the spores and growth of C. zanthoxyli and the induced resistance to leaf rust by Z. armatum were analyzed, and the possible defense mechanisms involved in SA induction were evaluated. The results showed that SA had no effect on C. zanthoxyli spore germination and growth; however, rust resistance was induced in Z. armatum. The optimal SA treatment concentration was 0.4 mg/ml, and the relative cure effect reached 44.56%. SA-induced disease resistance was evident for up to 10 days, while the optimal induction interval was 48 h after stimulation. Consistent with the induced resistance, H2O2, total protein, total phenol, and lignin concentrations and polyphenol oxidase (PPO), peroxidase (POD), phenylalanine ammonia lyase (PAL), superoxide dismutase (SOD), and catalase (CAT) activities were significantly increased with the SA treatment, whereas the malondialdehyde content was significantly decreased. In addition, exogenous SA promoted defense-related enzyme activities, including those of POD, CAT, and PAL, and increased H2O2, lignin, and endogenous SA contents. Furthermore, SA induced the expression of SA signaling pathway genes such as ZaPR1 and ZaNPR1, and silencing ZaPR1 enhanced the sensitivity of Z. armatum to leaf rust. Our results demonstrated that 0.4 mg/ml SA priming increased the activities of CAT, POD, and PAL, elevated the contents of H2O2, lignin, and endogenous SA, and upregulated the expression of the SA-related gene ZaPR1, thereby enhancing the resistance of Z. armatum to leaf rust.

Callus proliferation-induced hypoxic microenvironment decreases shoot regeneration competence in Arabidopsis

Plants are aerobic organisms that rely on molecular oxygen for respiratory energy production. Hypoxic conditions, with oxygen levels ranging between 1% and 5%, usually limit aerobic respiration and affect plant growth and development. Here, we demonstrate that the hypoxic microenvironment induced by active cell proliferation during the two-step plant regeneration process intrinsically represses the regeneration competence of the callus in Arabidopsis thaliana. We showed that hypoxia-repressed plant regeneration is mediated by the RELATED TO APETALA 2.12 (RAP2.12) protein, a member of the Ethylene Response Factor VII (ERF-VII) family. We found that the hypoxia-activated RAP2.12 protein promotes salicylic acid (SA) biosynthesis and defense responses, thereby inhibiting pluripotency acquisition and de novo shoot regeneration in calli. Molecular and genetic analyses revealed that RAP2.12 could bind directly to the SALICYLIC ACID INDUCTION DEFICIENT 2 (SID2) gene promoter and activate SA biosynthesis, repressing plant regeneration possibly via a PLETHORA (PLT)-dependent pathway. Consistently, the rap2.12 mutant calli exhibits enhanced shoot regeneration, which is impaired by SA treatment. Taken together, these findings uncover that the cell proliferation-dependent hypoxic microenvironment reduces cellular pluripotency and plant regeneration through the RAP2.12–SID2 module.

Effect of salicylic acid induction on germination, radicle length, and protein content in chickpea seedlings

Effect of salicylic acid induction on germination, radicle length, and protein content in chickpea seedlings

Promoter cloning and activities analysis of JmLFY, a key gene for flowering in Juglans mandshurica.

Juglans mandshurica (Manchurian walnut) is a precious timber and woody grain and oil species in Northeast China. The heterodichogamous characteristic phenomenon resulted in the non-synchronous flowering and development of male and female flowers, which limited the mating and the yield and quality of fruits. LFY is a core gene in the flowering regulatory networks, which has been cloned in J. mandshurica, and the function has also been verified preliminarily. In this study, the JmLFY promoter sequence with different lengths of 5'-deletion (pLFY1-pLFY6) were cloned and conducted bioinformatics analysis, the promoter activities were analyzed by detecting their driving activity to GUS gene in the tobacco plants that transformed with different promoter sequence stably or transiently. After that, the interaction between JmSOC1 and JmLFY gene promoter was also analyzed via yeast single-hybrid. The results showed that the promoter sequence contains core cis-acting elements essential for eukaryotic promoters, hormone response elements, defense- and stress-responsive elements, flowering-related elements, etc. Transgenic tobacco plants with pLFY1 were obtained by Agrobacterium infection using the pCAMBIA1301 expression vector, and the GUS gene driven by the JmLFY promoter was detected to express in the leaf, stem, flower, and root of the transformed tobacco plant, which indicated that the obtained JmLFY promoter had driving activity. GUS histochemical staining and enzyme activity detection showed that promoter fragments with different lengths had promoter activity and could respond to the induction of long photoperiod, low temperature, salicylic acid (SA), IAA, GA3, and methyl jasmonate (MeJA). The core regulatory region of JmLFY gene promoter in J. mandshurica was between -657 bp and -1,904 bp. Point-to-point validation of yeast single-hybrid confirmed the interaction between JmSOC1 and JmLFY gene promoter, which indicated that JmLFY gene is the downstream target of JmSOC1. These results reveal relevant factors affecting JmLFY gene expression and clarify the molecular mechanism of JmLFY gene regulation in the flower developmental partially, which will provide a theoretical basis for regulating the flowering time by regulating JmLFY gene expression in J. mandshurica.

Genome-Wide Identification and Analysis of the WRKY Gene Family in Asparagus officinalis.

In recent years, the related research of the WRKY gene family has been gradually promoted, which is mainly reflected in the aspects of environmental stress and hormone response. However, to make the study of the WRKY gene family more complete, we also need to focus on the whole-genome analysis and identification of the family. In previous studies, the whole WRKY gene family of Arabidopsis, legumes and other plants has been thoroughly studied. However, since the publication of Asparagus officinalis genome-wide data, there has never been an analysis of the whole WRKY gene family. To understand more broadly the function of the WRKY gene family, the whole genome and salt stress transcriptome data of asparagus were used for comprehensive analysis in this study, including WRKY gene family identification, phylogenetic tree construction, analysis of conserved mods and gene domains, extraction of cis-acting elements, intron/exon analysis, species collinearity analysis, and WRKY expression analysis under salt stress. The results showed that a total of 70 genes were selected and randomly distributed on 10 chromosomes and one undefined chromosome. According to the functional classification of Arabidopsis thaliana, the WRKY family of asparagus was divided into 11 subgroups (C1-C9, U1, U2). It is worth considering that the distribution rules of gene-conserved motifs, gene domains and introns/exons in the same subfamily are similar, which suggests that genes in the same subfamily may regulate similar physiological processes. In this study, 11 cis-acting elements of WRKY family were selected, among which auxin, gibberellin, abscisic acid, salicylic acid and other hormone-regulated induction elements were involved. In addition, environmental stress (such as drought stress and low-temperature response) also accounted for a large proportion. Interestingly, we analyzed a total of two tandem duplicate genes and 13 segmental duplication genes, suggesting that this is related to the amplification of the WRKY gene family. Transcriptome data analysis showed that WRKY family genes could regulate plant growth and development by up-regulating and down-regulating gene expression under salt stress. Volcanic maps showed that 3 and 15 AoWRKY genes were significantly up-regulated or down-regulated in NI&NI+S and AMF&AMF+S, respectively. These results provide a new way to analyze the evolution and function of the WRKY gene family, and can provide a reference for the production and research of asparagus.

Salicylic acid regulates phenolic acid biosynthesis via SmNPR1-SmTGA2/SmNPR4 modules in Salvia miltiorrhiza.

Phenolic acids are the main active ingredients in Salvia miltiorrhiza, which can be used for the treatment of many diseases, particularly cardiovascular diseases. It is known that salicylic acid (SA) can enhance phenolic acid content, but the molecular mechanism of its regulation is still unclear. The nonexpresser of PR genes 1 (NPR1) plays a positive role in the SA signaling pathway. In this study, we identified a SmNPR1 gene that responds to SA induction and systematically investigated its function. We found that SmNPR1 positively affected phenolic acid biosynthesis. Then, we identified a novel TGA transcription factor, SmTGA2, which interacts with SmNPR1. SmTGA2 positively regulates phenolic acid biosynthesis by directly up-regulating SmCYP98A14 expression. After double-gene transgenic analysis and other biochemical assays, it was found that SmNPR1 and SmTGA2 work synergistically to regulate phenolic acid biosynthesis. In addition, SmNPR4 forms a heterodimer with SmNPR1 to inhibit the function of SmNPR1, and SA can alleviate this effect. Collectively, these findings elucidate the molecular mechanism underlying the regulation of phenolic acid biosynthesis by SmNPR1-SmTGA2/SmNPR4 modules and provide novel insights into the study of the SA signaling pathway regulating plant secondary metabolism.

EDR1 associates with its homologs to synergistically regulate plant immunity in Arabidopsis

ENHANCED DISEASE RESISTANCE 1 (EDR1), a Raf-like mitogen-activated protein kinase (MAPK) kinase kinase (MAPKKK), is a negative regulator of resistance. There are three homologs, RAF3/4/5, of EDR1 in Arabidopsis. However, the roles of RAF3/4/5 in resistance and their functional link with EDR1 in plant immunity remain unclear. Here, we showed that the raf3/4/5 triple mutant displayed wild-type-like phenotypes to the powdery mildew pathogen Golovinomyces cichoracearum UCSC1 and the bacterial pathogen Pseudomonas syringae pv. tomato (Pto) DC3000. However, the edr1 raf3/4/5 quadruple mutant exhibited enhanced resistance to G. cichoracearum UCSC1 and Pto DC3000 compared to edr1. Consistently, MPK3/6 kinase activity was more highly activated in edr1 raf3/4/5 than that in edr1. Moreover, the enhanced resistance of edr1 raf3/4/5 required SALICYLIC ACID INDUCTION DEFICIENT 2 (SID2), an isochorismate synthase required for salicylic acid (SA) synthesis. Additionally, unlike EDR1, RAF3/4/5 weakly and indirectly associated with MKK4/5, and EDR1 was directly associated with RAF3/4/5. Taken together, these data indicate that EDR1 associates with RAF3/4/5, and they may function together to synergistically suppress MAPK cascades activation, which reveal the complexity and importance of Raf-like MAPKKKs in plant immunity regulation.

Cloning and Expression Analysis of <i>PeDIR19</i> Gene in Poplar

Dirigent ( DIR ) gene plays an important role in the formation of lignin and lignans by controlling the stereoselective coupling of pineperin alcohol monomers in plants during the polymerization of lignin and lignans. In this study, we take the ‘Nanlin 895’ ( Populus deltoides × P. euramericana  cv ‘Nanlin 895’) as the material, and clone the PeDIR19  gene. PeDIR19 , the whole length of 588 bp, has a length of 555 bp in ORF region and has only a length of 33 bp intron in the middle. PeDIR19 protein codes 185 amino acids and is a hydrophobic protein. It has four conservative motifs and has a highly conservative structural functional domain between 50 bp ~ 142 bp. PeDIR19  gene was found to have the highest homology with Populus trichocarpa   PtDIR19 ( Potri.001G009100 ), with only four bases differences. By phylogenetic analysis, PeDIR19  gene belongs to DIR -B/D subfamily. Through tissue specific expression analysis of ‘Nanlin 895’ with different growth stages, it was found that PeDIR19  gene expression level was the highest in the root during the tissue culture seedlings and in the stem during the pot seedlings. PeDIR19  gene had a strong response after 1 d under the induction of Yangpan Erbaojun ( Marssonina brunnea ). Under the induction of salicylic acid, methyl jasmonate and abscisic acid stress, PeDIR19  gene can strongly respond to the stress of methyl jasmonate and abscisic acid and weakly respond to the salicylic acid stress. This study provides a solid foundation for further study on the function identification of DIR  gene in the biosynthetic pathway of plant resistance.

The long non-coding RNA GhlncRNA149.1 improves cotton defense response to aphid damage as a positive regulator

Long non-coding RNAs (lncRNAs) play crucial roles in various plant biological regulatory processes and defense responses to all sorts of biotic and abiotic stresses. However, there are limited studies on the function and mechanism of lncRNAs in plant defense responses to phytophagous insects. In this research, a lncRNA GhlncRNA149.1 was identified from upland cotton (Gossypium hirsutum L.). Depending on the position relationship between lncRNA and mRNA, we identified CC-NBS-LRR family gene GhA01G0129 as the target gene of GhlncRNA149.1. Expression levels of GhlncRNA149.1 and its target gene GhA01G0129 were significantly down-regulated in cotton cotyledons upon cotton aphid infestation. The expression level of GhlncRNA149.1 was significantly down-regulated after the induction of methyl jasmonate and salicylic acid, whereas expression level of its target gene GhA01G0129 was significantly up-regulated. Overexpression of GhlncRNA149.1 in cotton cotyledons enhanced the tolerance of cotton plants to cotton aphid feeding. Expectedly, silencing GhlncRNA149.1 increased susceptibility of cotton plants to cotton aphids. The expression level of GhA01G0129, the target gene of GhlncRNA149.1, was significantly up-regulated in cotton plants overexpressing GhlncRNA149.1, and significantly down-regulated in cotton plants silencing GhlncRNA149.1. The activity and expression levels of antioxidant enzymes superoxide dismutase (SOD), peroxidase (POD) and catalase (CAT) were up-regulated in cotton plants transiently overexpressing GhlncRNA149.1, while opposite results were obtained in cotton plants silencing GhlncRNA149.1. In addition, similar results were obtained in the expression levels of phenylalanine ammonia-lyase (PAL), polyphenol oxidase (PPO), enhanced disease susceptibility 1 (EDS1) and non-expression of pathogenesis-related gene 1 (NPR1) in cotton plants transiently overexpressing GhlncRNA149.1 and silencing GhlncRNA149.1. These results collectively suggest that GhlncRNA149.1 improve the plant defense response to aphid attack, and thus has potential for enforcing cotton aphid prevention and control.

Root colonization by the endophytic fungus Piriformospora indica shortens the juvenile phase of Piper nigrum L. by fine tuning the floral promotion pathways.

Piriformospora indica, the mutualistic biotrophic root colonizing endosymbiotic fungus belonging to the order Sebacinales, offers host plants various benefits and enhances its growth and performance. The effect of colonization of P. indica in Piper nigrum L. cv. Panniyur1 on growth advantages, floral induction and evocation was investigated. Growth and yield benefits are credited to the alteration in the phytohormone levels fine-tuned by plants in response to the fungal colonization and perpetuation. The remarkable upregulation in the phytohormone levels, as estimated by LC- MS/MS and quantified by qRT-PCR, revealed the effectual contribution by the endophyte. qRT-PCR results revealed a significant shift in the expression of putative flowering regulatory genes in the photoperiod induction pathway (FLOWERING LOCUS T, LEAFY, APETALA1, AGAMOUS, SUPPRESSOR OF CONSTANS 1, GIGANTEA, PHYTOCHROMEA, and CRYPTOCHROME1) gibberellin biosynthetic pathway genes (GIBBERELLIN 20-OXIDASE2, GIBBERELLIN 2-OXIDASE, DELLA PROTEIN REPRESSOR OF GA1-3 1) autonomous (FLOWERING LOCUS C, FLOWERING LOCUS VE, FLOWERING LOCUS CA), and age pathway (SQUAMOSA PROMOTER LIKE9, APETALA2). The endophytic colonization had no effect on vernalization (FLOWERING LOCUS C) or biotic stress pathways (SALICYLIC ACID INDUCTION DEFICIENT 2, WRKY family transcription factor 22). The data suggest that P. nigrum responds positively to P. indica colonization, affecting preponement in floral induction as well as evocation, and thereby shortening the juvenile phase of the crop.

Tobacco necrosis virus A overcomes local cell death response in Nicotiana tabacum

AbstractTobacco necrosis virus A (TNVA) belongs to the genus Alphanecrovirus in the Tombusviridae family. Symptoms induced by TNVA include necrotic lesions on inoculated leaves in a wide host range. The necrotic cell death response to certain viruses is poorly understood. This study characterizes the mechanisms underlying the local cell death triggered by TNVA in Nicotiana tabacum, showing that it shares several components with the hypersensitive response (HR) mediated by resistance proteins. TNVA perception triggers the accumulation of local viral‐derived small interference RNA and the regulation of biological processes related to pathogen‐associated molecular pattern (PAMP)‐triggered immunity (PTI), including hydrogen peroxide accumulation, cell wall reinforcement, the activation of unfolded protein response and the induction of salicylic acid (SA) and SA‐dependent pathways. However, these antiviral defences do not prevent either local virus multiplication or systemic movement, leading to disease development. Nevertheless, SA‐deficient NahG tobacco plants challenged with TNVA showed an increase in virus accumulation on noninoculated leaves, which was correlated with the development of systemic necrosis, highlighting the role of SA signalling in TNVA‐induced defence. In addition, SA treatment enhanced the local defence response to TNVA infection and suppressed systemic necrosis in N. benthamiana. Taken together, our data suggest that TNVA induces an impaired plant defence response by modulating host factors to persist at low levels in distal tissues.

RETRACTED: Alternative oxidase is involved in leaf senescence via regulation of Salicylic acid accumulation in tomato

This article has been retracted: please see Elsevier Policy on Article Withdrawal (https://www.elsevier.com/locate/withdrawalpolicy). This article has been retracted at the request of the corresponding author, Yu Wen, and the Editor-in-Chief. The corresponding author requested retraction, due to multiple self-reported issues and breaches of scientific ethical standards. The Editor-in-Chief no longer has trust in the veracity of the findings in the paper and therefore decided to retract it. This paper was submitted to the journal by Yu Wen without the knowledge or permission of the co-author Dawei Zhang. Authors submitting to the journal warrant that the publication is approved by all authors. As Dawei Zhang did not agree to be a co-author on the paper, this is a clear breach of the submission requirements for the journal, as well a breach of scientific ethical standards. As well as the issue with the authorship, there are problems with the veracity of the data published in the article. Specifically, there is a duplication of Fig 1g and Fig 2a. The corresponding author Yu Wen has indicated that the relevant experiments in the article are inadequate and the conclusions cannot be characterized by physiological data alone, especially in the fourth figure where further relevant molecular experiments need to be designed to refine the conclusion.

Identification and Expression Analysis of Stilbene Synthase Genes in Arachis hypogaea in Response to Methyl Jasmonate and Salicylic Acid Induction

Stilbene synthase is an important enzyme of the phenylpropanoid pathway, regulating the production of several biologically active stilbenoids. These compounds have antioxidant, anti-inflammatory, and anti-cancer properties. However, the detailed characterization of stilbene synthase genes in Arachis hypogaea has not yet been performed. In this study, the comprehensive characterization of stilbene synthase genes in A. hypogaea was conducted, commencing with identification, phylogenetic analysis, and study of their expression in response to exogenous hormonal treatment. We identified and isolated five AhSTSs genes and recorded their expression pattern in peanut (BARD-479) in response to methyl jasmonate (MeJA) and salicylic acid (SA) treatment. The presence of Chal_sti_synt, ACP_syn_III, and FAE1_CUT1_rppA domains in all AhSTSs indicated their role in the biosynthesis of stilbene and lipid metabolism. Cis-regulatory element analysis indicated their role in light responsiveness, defense responses, regulation of seed development, plant growth, and development. Despite close structural and functional similarities, expression and correlational analysis suggested that these genes may have a specific role in peanut, as individual AhSTS exhibited differential expression upon hormonal treatment in a genotype dependent manner. Further studies on functional characterization involving the transcriptional regulation of AhSTSs can clearly explain the differential expression of stilbene synthase genes to hormonal treatment.

Cross-protection of oilseed rape against Verticillium longisporum by the non-aggressive lineage A1/D2

Verticillium longisporum is a soil-borne vascular pathogen of oilseed rape and other Brassica crops. The limited availability of chemical control measures against vascular pathogens calls for the exploration of control alternatives. Cross-protection may be conveyed by the use of non-pathogenic or non-aggressive isolates of potential pathogens as biocontrol agents. V. longisporum consists of three lineages that originated from three independent hybridization events of four haploid Verticillium parents. Previous pathogenicity tests in the greenhouse have shown that lineage A1/D2 is non-pathogenic or non-aggressive on different Brassica and non-Brassica hosts. Thus, the cross-protection potential of the A1/D2 lineage against an aggressive V. longisporum isolate in oilseed rape was tested. With root-dip inoculation, A1/D2 reduced disease symptoms when applied before or at the same time as the aggressive isolate. The induction of salicylic acid, a signal known to play a role in basal and cultivar-related resistance, was not involved in the biocontrol mechanism. The most practical and best feasible method of application as a seed coat failed to confirm the biocontrol effect of A1/D2 observed with root-dip inoculation. Confocal microscopy analysis revealed that seed coating led to insufficient A1/D2 hyphal establishment on the roots compared to root-dip inoculation, which may explain the lack of a biocontrol effect after seed coating and illustrates the importance of the application method for efficacy of a cross-protective biocontrol agent.

Salicylic Acid and Phytoalexin Induction by a Bacterium that Causes Halo Blight in Beans.

Pseudomonas savastanoi pv. phaseolicola is a bacterium that causes halo blight in beans. Different varieties of beans have hypersensitive resistance to specific races of P. savastanoi pv. phaseolicola. During hypersensitive resistance, also known as effector-triggered immunity (ETI), beans produce hormones that signal molecular processes to produce phytoalexins that are presumed to be antibiotic to bacteria. To shed light on hormone and phytoalexin production during immunity, we inoculated beans with virulent and avirulent races of P. savastanoi pv. phaseolicola. We then used mass spectrometry to measure the accumulation of salicylic acid (SA), the primary hormone that controls immunity in plants, and other hormones including jasmonate, methyljasmonate, indole-3-acetic acid, abscisic acid, cytokinin, gibberellic acid, and 1-aminocyclopropane-1-carboxylic acid. SA, but no other examined hormone, consistently increased at sites of infection to greater levels in resistant beans compared with susceptible beans at 4 days after inoculation. We then monitored 10 candidate bean phytoalexins. Daidzein, genistein, kievitone, phaseollin, phaseollidin, coumestrol, and resveratrol substantially increased alongside SA in resistant beans but not in susceptible beans. In vitro culture assays revealed that SA, daidzein, genistein, coumestrol, and resveratrol inhibited P. savastanoi pv. phaseolicola race 5 culture growth. These results demonstrate that these phytoalexins may be regulated by SA and work with SA during ETI to restrict bacterial replication. This is the first report of antibiotic activity for daidzein, genistein, and resveratrol to P. savastanoi pv. phaseolicola. These results improve our understanding of the mechanistic output of ETI toward this bacterial pathogen of beans.

The Two Classes of Ceramide Synthases Play Different Roles in Plant Immunity and Cell Death.

Ceramide synthases (CSs) produce ceramides from long-chain bases (LCBs). However, how CSs regulate immunity and cell death in Arabidopsis thaliana remains unclear. Here, we decipher the roles of two classes of CS, CSI (LAG1 HOMOLOG 2, LOH2) and CSII (LOH1/3), in these processes. The loh1-2 and loh1-1 loh3-1 mutants were resistant to the bacterial pathogen Pseudomonas syringae pv maculicola (Psm) DG3 and exhibited programmed cell death (PCD), along with increased LCBs and ceramides, at later stages. In loh1-2, the Psm resistance, PCD, and sphingolipid accumulation were mostly suppressed by inactivation of the lipase-like proteins ENHANCED DISEASE SUSCEPTIBILITY 1 (EDS1) and PHYTOALEXIN DEFICIENT 4 (PAD4), and partly suppressed by loss of SALICYLIC ACID INDUCTION DEFICIENT 2 (SID2). The LOH1 inhibitor fumonisin B1 (FB1) triggered EDS1/PAD4-independent LCB accumulation, and EDS1/PAD4-dependent cell death, resistance to Psm, and C16 Cer accumulation. Loss of LOH2 enhances FB1-, and sphinganine-induced PCD, indicating that CSI negatively regulates the signaling triggered by CSII inhibition. Like Cer, LCBs mediate cell death and immunity signaling, partly through the EDS1/PAD4 pathway. Our results show that the two classes of ceramide synthases differentially regulate EDS1/PAD4-dependent PCD and immunity via subtle control of LCBs and Cers in Arabidopsis.

The Plant Defense Signal Salicylic Acid Activates the RpfB-Dependent Quorum Sensing Signal Turnover via Altering the Culture and Cytoplasmic pH in the Phytopathogen Xanthomonas campestris.

ABSTRACTPlant colonization by phytopathogens is a very complex process in which numerous factors are involved. Upon infection by phytopathogens, plants produce salicylic acid (SA) that triggers gene expression within the plant to counter the invading pathogens. The present study demonstrated that SA signal also directly acts on the quorum-sensing (QS) system of the invading pathogen Xanthomonas campestris pv. campestris to affect its virulence by inducing turnover of the diffusible signaling factor (DSF) family QS signal. First, Xanthomonas campestris pv. campestris infection induces SA biosynthesis in the cabbage host plant. SA cannot be degraded by Xanthomonas campestris pv. campestris during culturing. Exogenous addition of SA or endogenous production of SA induces DSF signal turnover during late growth phase of Xanthomonas campestris pv. campestris in XYS medium that mimics plant vascular environments. Further, the DSF turnover gene rpfB is required for SA induction of DSF turnover. However, SA does not affect the expression of rpfB and DSF biosynthesis gene rpfF at the transcriptional level. SA induction of DSF turnover only occurs under acidic conditions in XYS medium. Furthermore, addition of SA to XYS medium significantly increased both culture and cytoplasmic pH. Increased cytoplasmic pH induced DSF turnover in a rpfB-dependent manner. In vitro RpfB-dependent DSF turnover activity increased when pH increased from 6 to 8. SA exposure did not affect the RpfB-dependent DSF turnover in vitro. Finally, SA-treated Xanthomonas campestris pv. campestris strain exhibited enhanced virulence when inoculated on cabbage. These results provide new insight into the roles of SA in host plants and the molecular interactions between Xanthomonas campestris pv. campestris and cruciferous plants.

Specialist Aphid Feeding Causes Local Activation of Salicylic and Jasmonic Acid Signaling in Arabidopsis Veins.

Aphids, the phloem sap feeders, probe into leaf tissues and activate a complex network of plant defense responses. Phytohormonal signaling plays a major role in this network; however, the dynamics of the signal spreading is yet to be clarified. Despite the growing knowledge about transcriptomic changes upon infestation, results often differ due to sampling, varying strongly between the tissues collected at the single feeding site, individual leaves, pooled infested leaves, or whole plant rosettes. This study focuses on activation of salicylic acid (SA) and jasmonic acid (JA) signals in Arabidopsis leaves during infestation by cabbage aphid (Brevicoryne brassicae) in high spatio-temporal resolution. We used genetically encoded fluorescent biosensors, histochemistry, and quantitative reverse transcription-PCR to precisely map activation of distinct branches of phytohormonal signaling. We found a rapid induction of SA and JA signaling markers in cells surrounding stylet puncture, colocalizing with callose deposition. For both PR1 and JAZ10, we detected activation at 24 h postinfestation (hpi), increasing and spreading along the veins until 72 hpi and, to a lesser extent, within the epidermal pavement cells. The SA signaling wave appeared in parallel with JA-associated signaling and continued to increase in time. Our results first show a local activation of SA- and JA-related responses after stylet penetration of Arabidopsis leaves and bring a detailed insight into the spatio-temporal complexity of plant defense activation during specialist aphid attack.[Formula: see text] Copyright © 2022 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.