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.

Negative modulation of SA signaling components by the capsid protein of tobacco mosaic virus is required for viral long-distance movement.

An important aspect of plant-virus interaction is the way viruses dynamically move over long distances and how plant immunity modulates viral systemic movement. Salicylic acid (SA), a well-characterized hormone responsible for immune responses against virus, is activated through different transcription factors including TGA and WRKY. In tobamoviruses, evidence suggests that capsid protein (CP) is required for long-distance movement, although its precise role has not been fully characterized yet. Previously, we showed that the CP of Tobacco Mosaic Virus (TMV)-Cg negatively modulates the SA-mediated defense. In this study, we analyzed the impact of SA-defense mechanism on the long-distance transport of a truncated version of TMV (TMV ∆CP virus) that cannot move to systemic tissues. The study showed that the negative modulation of NPR1 and TGA10 factors allows the long-distance transport of TMV ∆CP virus. Moreover, we observed that the stabilization of DELLA proteins promotes TMV ∆CP systemic movement. We also characterized a group of genes, part of a network modulated by CP, involved in TMV ∆CP long-distance transport. Altogether, our results indicate that CP-mediated downregulation of SA signaling pathway is required for the virus systemic movement, and this role of CP may be linked to its ability to stabilize DELLA proteins.

More stories to tell: NONEXPRESSOR OF PATHOGENESIS-RELATED GENES1, a salicylic acid receptor.

Salicylic acid (SA) plays pivotal role in plant defense against biotrophic and hemibiotrophic pathogens. Tremendous progress has been made in the field of SA biosynthesis and SA signaling pathways over the past three decades. Among the key immune players in SA signaling pathway, NONEXPRESSOR OF PATHOGENESIS-RELATED GENES1 (NPR1) functions as a master regulator of SA-mediated plant defense. The function of NPR1 as an SA receptor has been controversial; however, after years of arguments among several laboratories, NPR1 has finally been proven as one of the SA receptors. The function of NPR1 is strictly regulated via post-translational modifications and transcriptional regulation that were recently found. More recent advances in NPR1 biology, including novel functions of NPR1 and the structure of SA receptor proteins, have brought this field forward immensely. Therefore, based on these recent discoveries, this review acts to provide a full picture of how NPR1 functions in plant immunity and how NPR1 gene and NPR1 protein are regulated at multiple levels. Finally, we also discuss potential challenges in future studies of SA signaling pathway.

R2R3-MYB Transcription Factor MdMYB73 Confers Increased Resistance to the Fungal Pathogen Botryosphaeria dothidea in Apples via the Salicylic Acid Pathway.

MYB transcription factors (TFs) participate in many biological processes. However, the molecular mechanisms by which MYB TFs affect plant resistance to apple ring rot remain poorly understood. Here, the R2R3-MYB gene MdMYB73 was cloned from "Royal Gala" apples and functionally characterized as a positive regulator of the defense response to Botryosphaeria dothidea. qRT-PCR and GUS staining demonstrated that MdMYB73 was strongly induced in apple fruits and transgenic calli after inoculation with B. dothidea. MdMYB73 overexpression improved resistance to B. dothidea in apple calli and fruits, while MdMYB73 suppression weakened. Increased resistance to B. dothidea was also observed in MdMYB73-expressing Arabidopsis thaliana. Interestingly, salicylic acid (SA) contents and the expression levels of genes related with SA synthesis and signaling were greater in MdMYB73-overexpressing plant materials compared to wild-type controls after inoculation, suggesting that MdMYB73 might enhance resistance to B. dothidea via the SA pathway. Finally, we discovered that MdMYB73 interacts with MdWRKY31, a positive regulator of B. dothidea. Together, MdWRKY31 and MdMYB73 enhanced B. dothidea resistance in apples. Our results clarify the mechanisms by which MdMYB73 improves resistance to B. dothidea and suggest that resistance may be affected by regulating the SA pathway.

Formation of NPR1 Condensates Promotes Cell Survival during the Plant Immune Response.

In plants, pathogen effector-triggered immunity (ETI) often leads to programmed cell death, which is restricted by NPR1, an activator of systemic acquired resistance. However, the biochemical activities of NPR1 enabling it to promote defense and restrict cell death remain unclear. Here we show that NPR1 promotes cell survival by targeting substrates for ubiquitination and degradation through formation of salicylic acid-induced NPR1 condensates (SINCs). SINCs are enriched with stress response proteins, including nucleotide-binding leucine-rich repeat immune receptors, oxidative and DNA damage response proteins, and protein quality control machineries. Transition of NPR1 into condensates is required for formation of the NPR1-Cullin 3 E3 ligase complex to ubiquitinate SINC-localized substrates, such as EDS1 and specific WRKY transcription factors, and promote cell survival during ETI. Our analysis of SINCs suggests that NPR1 is centrally integrated into the cell death or survival decisions in plant immunity by modulating multiple stress-responsive processes in this quasi-organelle.

A Defense Pathway Linking Plasma Membrane and Chloroplasts and Co-opted by Pathogens.

Chloroplasts are crucial players in the activation of defensive hormonal responses during plant-pathogen interactions. Here, we show that a plant virus-encoded protein re-localizes from the plasma membrane to chloroplasts upon activation of plant defense, interfering with the chloroplast-dependent anti-viral salicylic acid (SA) biosynthesis. Strikingly, we have found that plant pathogens from different kingdoms seem to have convergently evolved to target chloroplasts and impair SA-dependent defenses following an association with membranes, which relies on the co-existence of two subcellular targeting signals, an N-myristoylation site and a chloroplast transit peptide. This pattern is also present in plant proteins, at least one of which conversely activates SA defenses from the chloroplast. Taken together, our results suggest that a pathway linking plasma membrane to chloroplasts and activating defense exists in plants and that such pathway has been co-opted by plant pathogens during host-pathogen co-evolution to promote virulence through suppression of SA responses.

Salicylic acid: transport and long-distance immune signaling.

The small phenolic compound salicylic acid (SA) is a phytohormone that regulates many biological processes, although it is most well-known for its role in plant defense. SA is an important regulator of systemic acquired resistance (SAR), a type of systemic immunity that protects uninfected parts of the plant against secondary infections by a broad spectrum of pathogens. SAR involves the generation of mobile signal(s) at the primary infection site, which translocate to distal uninfected portions and activate systemic disease resistance. Although, SA was considered to not constitute the mobile SAR signal, it is preferentially transported from pathogen-infected to uninfected parts via the apoplast. Further investigations have revealed that distal transport of SA is indeed essential for SAR. The apoplastic SA transport is regulated by the transpirational pull and partitioning of SA between the symplast and cuticle.

Highly Selective and Sensitive Detection of Biogenic Defense Phytohormone Salicylic Acid in Living Cells and Plants Using a Novel and Viable Rhodamine-Functionalized Fluorescent Probe.

Detecting plant-derived signal molecules using fluorescent probes is a key topic and a huge challenge for scientists. Salicylic acid (SA), a vital plant-derived defense hormone, can activate global transcriptional reprogramming to systemically express a network of prominent pathogenesis-related proteins against invasive microorganisms. This strategy is called systemic acquired resistance (SAR). Therefore, monitoring the dynamic fluctuations of SA in subcellular microenvironments can advance our understanding of different physiological and pathological functions during the SA-induced SAR mechanism, thus benefiting the discovery and development of novel immune activators that contribute to crop protection. Here, detection of signaling molecule SA in plant callus tissues was first reported and conducted by a simple non-fluorescent rhodamine-tagged architecture bearing a flexible 2-amino-N,N-dimethylacetamide pattern. This study can markedly advance and promote the usage of fluorescent SA probes for distinguishing SA in the plant kingdom.

Biosynthesis and Regulation of Salicylic Acid and N-Hydroxypipecolic Acid in Plant Immunity.

Salicylic acid (SA) has long been known to be essential for basal defense and systemic acquired resistance (SAR). N-Hydroxypipecolic acid (NHP), a recently discovered plant metabolite, also plays a key role in SAR and to a lesser extent in basal resistance. Following pathogen infection, levels of both compounds are dramatically increased. Analysis of SA- or SAR-deficient mutants has uncovered how SA and NHP are biosynthesized. The completion of the SA and NHP biosynthetic pathways in Arabidopsis allowed better understanding of how they are regulated. In this review, we discuss recent progress on SA and NHP biosynthesis and their regulation in plant immunity.

Early Pep-13-induced immune responses are SERK3A/B-dependent in potato.

Potato plants treated with the pathogen-associated molecular pattern Pep-13 mount salicylic acid- and jasmonic acid-dependent defense responses, leading to enhanced resistance against Phytophthora infestans, the causal agent of late blight disease. Recognition of Pep-13 is assumed to occur by binding to a yet unknown plasma membrane-localized receptor kinase. The potato genes annotated to encode the co-receptor BAK1, StSERK3A and StSERK3B, are activated in response to Pep-13 treatment. Transgenic RNAi-potato plants with reduced expression of both SERK3A and SERK3B were generated. In response to Pep-13 treatment, the formation of reactive oxygen species and MAP kinase activation, observed in wild type plants, is highly reduced in StSERK3A/B-RNAi plants, suggesting that StSERK3A/B are required for perception of Pep-13 in potato. In contrast, defense gene expression is induced by Pep-13 in both control and StSERK3A/B-depleted plants. Altered morphology of StSERK3A/B-RNAi plants correlates with major shifts in metabolism, as determined by untargeted metabolite profiling. Enhanced levels of hydroxycinnamic acid amides, typical phytoalexins of potato, in StSERK3A/B-RNAi plants are accompanied by significantly decreased levels of flavonoids and steroidal glycoalkaloids. Thus, altered metabolism in StSERK3A/B-RNAi plants correlates with the ability of StSERK3A/B-depleted plants to mount defense, despite highly decreased early immune responses.

Systemic acquired resistance networks amplify airborne defense cues.

Salicylic acid (SA)-mediated innate immune responses are activated in plants perceiving volatile monoterpenes. Here, we show that monoterpene-associated responses are propagated in feed-forward loops involving the systemic acquired resistance (SAR) signaling components pipecolic acid, glycerol-3-phosphate, and LEGUME LECTIN-LIKE PROTEIN1 (LLP1). In this cascade, LLP1 forms a key regulatory unit in both within-plant and between-plant propagation of immunity. The data integrate molecular components of SAR into systemic signaling networks that are separate from conventional, SA-associated innate immune mechanisms. These networks are central to plant-to-plant propagation of immunity, potentially raising SAR to the population level. In this process, monoterpenes act as microbe-inducible plant volatiles, which as part of plant-derived volatile blends have the potential to promote the generation of a wave of innate immune signaling within canopies or plant stands. Hence, plant-to-plant propagation of SAR holds significant potential to fortify future durable crop protection strategies following a single volatile trigger.

Gene networks underlying the early regulation of Paraburkholderia phytofirmans PsJN induced systemic resistance in Arabidopsis.

Plant defense responses to biotic stresses are complex biological processes, all governed by sophisticated molecular regulations. Induced systemic resistance (ISR) is one of these defense mechanisms where beneficial bacteria or fungi prime plants to resist pathogens or pest attacks. In ISR, the defense arsenal in plants remains dormant and it is only triggered by an infection, allowing a better allocation of plant resources. Our group recently described that the well-known beneficial bacterium Paraburkholderia phytofirmans PsJN is able to induce Arabidopsis thaliana resistance to Pseudomonas syringae pv. tomato (Pst) DC3000 through ISR, and that ethylene, jasmonate and salicylic acid are involved in this protection. Nevertheless, the molecular networks governing this beneficial interaction remain unknown. To tackle this issue, we analyzed the temporal changes in the transcriptome of PsJN-inoculated plants before and after being infected with Pst DC3000. These data were used to perform a gene network analysis to identify highly connected transcription factors. Before the pathogen challenge, the strain PsJN regulated 405 genes (corresponding to 1.8% of the analyzed genome). PsJN-inoculated plants presented a faster and stronger transcriptional response at 1-hour post infection (hpi) compared with the non-inoculated plants, which presented the highest transcriptional changes at 24 hpi. A principal component analysis showed that PsJN-induced plant responses to the pathogen could be differentiated from those induced by the pathogen itself. Forty-eight transcription factors were regulated by PsJN at 1 hpi, and a system biology analysis revealed a network with four clusters. Within these clusters LHY, WRKY28, MYB31 and RRTF1 are highly connected transcription factors, which could act as hub regulators in this interaction. Concordantly with our previous results, these clusters are related to jasmonate, ethylene, salicylic, acid and ROS pathways. These results indicate that a rapid and specific response of PsJN-inoculated plants to the virulent DC3000 strain could be the pivotal element in the protection mechanism.

AtNPR4 from Arabidopsis thaliana: expression, purification, crystallization and crystallographic analysis.

Salicylic acid (SA) is an important phytohormone that is involved in the regulation of plant defence, growth and development. A large number of proteins have been shown to have the ability to interact with SA, and NPR4 has been demonstrated to be a receptor of SA that plays significant roles in the innate immune response of plants. In this study, Spodoptera frugiperda (Sf9) cells were used to express full-length AtNPR4 from Arabidopsis thaliana. To facilitate crystallization, T4 lysozyme (T4L) was added to the N-terminus of the AtNPR4 protein. The recombinant T4L-AtNPR4 protein was expressed, purified and crystallized using the sitting-drop and hanging-drop vapour-diffusion methods. The T4L-AtNPR4 crystals have symmetry consistent with space group C2, with unit-cell parameters a = 93.7, b = 85.8, c = 88.2 Å, ß = 90° and one molecule per asymmetric unit. The best crystal diffracted to a resolution of 2.75 Å. Structure determination is in progress.

A kiwellin disarms the metabolic activity of a secreted fungal virulence factor.

Fungi-induced plant diseases affect global food security and plant ecology. The biotrophic fungus Ustilago maydis causes smut disease in maize (Zea mays) plants by secreting numerous virulence effectors that reprogram plant metabolism and immune responses1,2. The secreted fungal chorismate mutase Cmu1 presumably affects biosynthesis of the plant immune signal salicylic acid by channelling chorismate into the phenylpropanoid pathway3. Here we show that one of the 20 maize-encoded kiwellins (ZmKWL1) specifically blocks the catalytic activity of Cmu1. ZmKWL1 hinders substrate access to the active site of Cmu1 through intimate interactions involving structural features that are specific to fungal Cmu1 orthologues. Phylogenetic analysis suggests that plant kiwellins have a versatile scaffold that can specifically counteract pathogen effectors such as Cmu1. We reveal the biological activity of a member of the kiwellin family, a widely conserved group of proteins that have previously been recognized only as important human allergens.

Long-Lasting Primed State in Maize Plants: Salicylic Acid and Steroid Signaling Pathways as Key Players in the Early Activation of Immune Responses in Silks.

In the present study, we investigated the induced systemic resistance (ISR) activated by the beneficial fungus Trichoderma atroviride in maize plants, and the early immunological responses triggered after challenge with the ear rot pathogen Fusarium verticillioides. By transcriptional analysis, we were able to identify the gene core set specifically modulated in silks of maize plants expressing ISR. Our results showed that the main transcriptional reprogramming falls into genes involved in five main functional categories: cell structure or cell wall, amino acid and protein metabolism, stress responses, signaling, and transport. Among these ISR-related genes, it is important to highlight novel findings regarding hormone metabolism and signaling. The expression of hormone-dependent genes was in good agreement with the abscisic acid, jasmonic acid, and salicylic acid (SA) levels detected in the plants under study. The experimental design allowed the identification of novel regulatory elements related to a heightened state of defense in silks and suggests that steroids and SA are central components of a master regulatory network controlling the immunity of silks during ISR. The results presented also provide evidence about the molecular mechanisms used by maize silks against F. verticillioides to counteract pathogenic development and host invasion, including pathogenesis-related genes, plant cell-wall reinforcement, fungal cell-wall-degrading enzymes and secondary metabolism.

New salicylic acid and pyroglutamic acid conjugated derivatives confer protection to bread wheat against Zymoseptoria tritici.

BACKGROUND: To promote sustainable agriculture and healthy food, research that contributes towards a new generation of eco-friendly phytosanitary compounds is increasingly encouraged. The plant hormone salicylic acid (SA) is known for its ability to induce resistance in plants against a wide range of pathogens, whereas pyroglutamic acid (PGA), a constrained analogue of γ-aminobutyric acid, has never been studied in the context of plant protection. RESULTS: The present study investigated for the first time the protection efficacy of SA and PGA and five new conjugated derivatives against Zymoseptoria tritici, the main pathogen in wheat crops. SA and four derivatives showed significant disease severity reductions in planta (up to 49%). In vitro assays revealed that some molecules, including SA, displayed a small direct antifungal activity, whereas others, such as PGA, showed no effect. This finding suggests that, especially for molecules without any direct activity, the mode of action relies mainly on the induction of plant resistance. CONCLUSION: Further investigations are needed to identify the defence pathways involved in plant resistance mechanisms elicited or primed by the molecules. The manufacture of these products was easily achieved on a scale of tens of grams of raw materials, and is easily scalable. The synthetic pathway is simple, short and inexpensive. For all of these reasons, the production of the target molecules is attractive for producers, whereas the prospect of a generation of non-polluting compounds with lasting efficiency against Z. tritici in wheat comes at a key moment for the sustainability of agriculture. © 2018 Society of Chemical Industry.

Transcriptome analysis of an incompatible Persea americana-Phytophthora cinnamomi interaction reveals the involvement of SA- and JA-pathways in a successful defense response.

Phytophthora cinnamomi Rands (Pc) is a hemibiotrophic oomycete and the causal agent of Phytophthora root rot (PRR) of the commercially important fruit crop avocado (Persea americana Mill.). Plant defense against pathogens is modulated by phytohormone signaling pathways such as salicylic acid (SA), jasmonic acid (JA), ethylene (ET), auxin and abscisic acid. The role of specific signaling pathways induced and regulated during hemibiotroph-plant interactions has been widely debated. Some studies report SA mediated defense while others hypothesize that JA responses restrict the spread of pathogens. This study aimed to identify the role of SA- and JA- associated genes in the defense strategy of a resistant avocado rootstock, Dusa in response to Pc infection. Transcripts associated with SA-mediated defense pathways and lignin biosynthesis were upregulated at 6 hours post-inoculation (hpi). Results suggest that auxin, reactive oxygen species (ROS) and Ca2+ signaling was also important during this early time point, while JA signaling was absent. Both SA and JA defense responses were shown to play a role during defense at 18 hpi. Induction of genes associated with ROS detoxification and cell wall digestion (ß-1-3-glucanase) was also observed. Most genes induced at 24 hpi were linked to JA responses. Other processes at play in avocado at 24 hpi include cell wall strengthening, the formation of phenolics and induction of arabinogalactan, a gene linked to Pc zoospore immobility. This study represents the first transcriptome wide analysis of a resistant avocado rootstock treated with SA and JA compared to Pc infection. The results provide evidence of a biphasic defense response against the hemibiotroph, which initially involves SA-mediated gene expression followed by the enrichment of JA-mediated defense from 18 to 24 hpi. Genes and molecular pathways linked to Pc resistance are highlighted and may serve as future targets for manipulation in the development of PRR resistant avocado rootstocks.

CRISPR/Cas9-Mediated Immunity in Plants Against Pathogens.

Global crop production is highly threatened due to pathogen invasion. The huge quantity of pesticides application, although harmful to the environment and human health, is carried out to prevent the crop losses worldwide, every year. Therefore, understanding the molecular mechanisms of pathogenicity and plant resistance against pathogen is important. The resistance against pathogens is regulated by three important phytohormones viz. salicylic acid (SA), jasmonic acid (JA) and ethylene (ET). Here we review possible role of CRISPR technology to understand the plant pathogenicity by mutating genes responsible for pathogen invasion or up-regulating the phytohormones genes or resistant genes. Thus hormone biosynthesis genes, receptor and feeding genes of pathogens could be important targets for modifications using CRISPR/Cas9 following multiplexing tool box strategy in order to edit multiple genes simultaneously to produce super plants. Here we put forward our idea thatthe genes would be either mutated in case of plant receptor protein targets of pathogens or up-regulation of resistant genes or hormone biosynthesis genes will be better choice for resistance against pathogens.

The highly buffered Arabidopsis immune signaling network conceals the functions of its components.

Plant immunity protects plants from numerous potentially pathogenic microbes. The biological network that controls plant inducible immunity must function effectively even when network components are targeted and disabled by pathogen effectors. Network buffering could confer this resilience by allowing different parts of the network to compensate for loss of one another's functions. Networks rich in buffering rely on interactions within the network, but these mechanisms are difficult to study by simple genetic means. Through a network reconstitution strategy, in which we disassemble and stepwise reassemble the plant immune network that mediates Pattern-Triggered-Immunity, we have resolved systems-level regulatory mechanisms underlying the Arabidopsis transcriptome response to the immune stimulant flagellin-22 (flg22). These mechanisms show widespread evidence of interactions among major sub-networks-we call these sectors-in the flg22-responsive transcriptome. Many of these interactions result in network buffering. Resolved regulatory mechanisms show unexpected patterns for how the jasmonate (JA), ethylene (ET), phytoalexin-deficient 4 (PAD4), and salicylate (SA) signaling sectors control the transcriptional response to flg22. We demonstrate that many of the regulatory mechanisms we resolved are not detectable by the traditional genetic approach of single-gene null-mutant analysis. Similar to potential pathogenic perturbations, null-mutant effects on immune signaling can be buffered by the network.