Systemic acquired resistance-associated transport and metabolic regulation of salicylic acid and glycerol-3-phosphate.

Systemic acquired resistance (SAR), a type of long-distance immunity in plants, provides long-lasting resistance to a broad spectrum of pathogens. SAR is thought to involve the rapid generation and systemic transport of a mobile signal that prepares systemic parts of the plant to better resist future infections. Exploration of the molecular mechanisms underlying SAR have identified multiple mobile regulators of SAR in the last few decades. Examination of the relationship among several of these seemingly unrelated molecules depicts a forked pathway comprising at least two branches of equal importance to SAR. One branch is regulated by the plant hormone salicylic acid (SA), and the other culminates (based on current knowledge) with the phosphorylated sugar derivative, glycerol-3-phosphate (G3P). This review summarizes the activities that contribute to pathogen-responsive generation of SA and G3P and the components that regulate their systemic transport during SAR.

Modulation of GmFAD3 expression alters abiotic stress responses in soybean.

KEY MESSAGE: This study focused on enhancing resilience of soybean crops to drought and salinity stresses by overexpression of GmFAD3A gene, which plays an important role in modulating membrane fluidity and ultimately influence plants response to various abiotic stresses. Fatty acid desaturases (FADs) are a class of enzymes that mediate desaturation of fatty acids by introducing double bonds. They play an important role in modulating membrane fluidity in response to various abiotic stresses. However, a comprehensive analysis of GmFAD3 in drought and salinity stress tolerance in soybean is lacking. We used bean pod mottle virus (BPMV)-based vector for achieving rapid and efficient overexpression as well as silencing of Omega-3 Fatty Acid Desaturase gene from Glycine max (GmFAD3) to assess the functional role of GmFAD3 in abiotic stress responses in soybean. Higher levels of recombinant BPMV-GmFAD3A transcripts were detected in overexpressing soybean plants. Overexpression of GmFAD3A in soybean resulted in increased levels of jasmonic acid and higher expression of GmWRKY54 as compared to mock-inoculated, vector-infected and FAD3-silenced soybean plants under drought and salinity stress conditions. The GmFAD3A-overexpressing plants showed higher levels of chlorophyll content, efficient photosystem-II, relative water content, transpiration rate, stomatal conductance, proline content and also cooler canopy under drought and salinity stress conditions as compared to mock-inoculated, vector-infected and FAD3-silenced soybean plants. Results from the current study revealed that GmFAD3A-overexpressing soybean plants exhibited tolerance to drought and salinity stresses. However, soybean plants silenced for GmFAD3 were vulnerable to drought and salinity stresses.

Phased small RNA-mediated systemic signaling in plants.

Systemic acquired resistance (SAR) involves the generation of systemically transported signal that arms distal plant parts against secondary infections. We show that two phased 21-nucleotide (nt) trans-acting small interfering RNA3a RNAs (tasi-RNA) derived from TAS3a and synthesized within 3 hours of pathogen infection are the early mobile signal in SAR. TAS3a undergoes alternate polyadenylation, resulting in the generation of 555- and 367-nt transcripts. The 555-nt transcripts likely serves as the sole precursor for tasi-RNAs D7 and D8, which cleave Auxin response factors (ARF) 2, 3, and 4 to induce SAR. Conversely, increased expression of ARF3 represses SAR. Knockout mutations in TAS3a or RNA silencing components required for tasi-RNA biogenesis compromise SAR without altering levels of known SAR-inducing chemicals. Both tasi-ARFs and the 367-nt transcripts are mobile and transported via plasmodesmata. Together, we show that tasi-ARFs are the early mobile signal in SAR.

Mobile signals in systemic acquired resistance.

Plants possess a unique form of broad-spectrum long-distance immunity termed systemic acquired resistance (SAR). SAR involves the rapid generation of mobile signal(s) in response to localized microbial infection, which transport to the distal tissue and 'prime' them against future infections by related and unrelated pathogens. Several SAR-inducing chemicals that could be classified as the potential mobile signal have been identified. Many of these function in a bifurcate pathway with both branches being equally essential for SAR induction. This review reflects on the potential candidacy of the known SAR inducers as mobile signal(s) based on historical knowledge of the SAR signal and recent advances in the SAR signaling pathway.

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.

The plant cuticle regulates apoplastic transport of salicylic acid during systemic acquired resistance.

The plant cuticle is often considered a passive barrier from the environment. We show that the cuticle regulates active transport of the defense hormone salicylic acid (SA). SA, an important regulator of systemic acquired resistance (SAR), is preferentially transported from pathogen-infected to uninfected parts via the apoplast. Apoplastic accumulation of SA, which precedes its accumulation in the cytosol, is driven by the pH gradient and deprotonation of SA. In cuticle-defective mutants, increased transpiration and reduced water potential preferentially routes SA to cuticle wax rather than to the apoplast. This results in defective long-distance transport of SA, which in turn impairs distal accumulation of the SAR-inducer pipecolic acid. High humidity reduces transpiration to restore systemic SA transport and, thereby, SAR in cuticle-defective mutants. Together, our results demonstrate that long-distance mobility of SA is essential for SAR and that partitioning of SA between the symplast and cuticle is regulated by transpiration.

A transcriptional regulatory network of Rsv3-mediated extreme resistance against Soybean mosaic virus.

Resistance genes are an effective means for disease control in plants. They predominantly function by inducing a hypersensitive reaction, which results in localized cell death restricting pathogen spread. Some resistance genes elicit an atypical response, termed extreme resistance, where resistance is not associated with a hypersensitive reaction and its standard defense responses. Unlike hypersensitive reaction, the molecular regulatory mechanism(s) underlying extreme resistance is largely unexplored. One of the few known, naturally occurring, instances of extreme resistance is resistance derived from the soybean Rsv3 gene, which confers resistance against the most virulent Soybean mosaic virus strains. To discern the regulatory mechanism underlying Rsv3-mediated extreme resistance, we generated a gene regulatory network using transcriptomic data from time course comparisons of Soybean mosaic virus-G7-inoculated resistant (L29, Rsv3-genotype) and susceptible (Williams82, rsv3-genotype) soybean cultivars. Our results show Rsv3 begins mounting a defense by 6 hpi via a complex phytohormone network, where abscisic acid, cytokinin, jasmonic acid, and salicylic acid pathways are suppressed. We identified putative regulatory interactions between transcription factors and genes in phytohormone regulatory pathways, which is consistent with the demonstrated involvement of these pathways in Rsv3-mediated resistance. One such transcription factor identified as a putative transcriptional regulator was MYC2 encoded by Glyma.07G051500. Known as a master regulator of abscisic acid and jasmonic acid signaling, MYC2 specifically recognizes the G-box motif ("CACGTG"), which was significantly enriched in our data among differentially expressed genes implicated in abscisic acid- and jasmonic acid-related activities. This suggests an important role for Glyma.07G051500 in abscisic acid- and jasmonic acid-derived defense signaling in Rsv3. Resultantly, the findings from our network offer insights into genes and biological pathways underlying the molecular defense mechanism of Rsv3-mediated extreme resistance against Soybean mosaic virus. The computational pipeline used to reconstruct the gene regulatory network in this study is freely available at https://github.com/LiLabAtVT/rsv3-network.

Lipid-Modulated Trafficking in Plants.

Scientific progress in recent years has significantly unraveled several unique structural and functional aspects of the plasmodesmata (PD), such as demonstrating the presence of detergent-insoluble membrane microdomains enriched in sterols and sphingolipids. A recent study now shows that one of the sphingolipids, t18:0 phytoshinganine, binds to PD localizing protein 5 (PDLP5) and increases retention of PDLP5 at PD, which is known to be associated with reduced PD permeability. The dynamic interaction between lipids and PD-associated proteins assemble yet another piece of the PD puzzle.

Glycerol-3-phosphate mediates rhizobia-induced systemic signaling in soybean.

Glycerol-3-phosphate (G3P) is a well-known mobile regulator of systemic acquired resistance (SAR), which provides broad spectrum systemic immunity in response to localized foliar pathogenic infections. We show that G3P-derived foliar immunity is also activated in response to genetically-regulated incompatible interactions with nitrogen-fixing bacteria. Using gene knock-down we show that G3P is essential for strain-specific exclusion of non-desirable root-nodulating bacteria and the associated foliar pathogen immunity in soybean. Grafting studies show that while recognition of rhizobium incompatibility is root driven, bacterial exclusion requires G3P biosynthesis in the shoot. Biochemical analyses support shoot-to-root transport of G3P during incompatible rhizobia interaction. We describe a root-shoot-root signaling mechanism which simultaneously enables the plant to exclude non-desirable nitrogen-fixing rhizobia in the root and pathogenic microbes in the shoot.

The analogous and opposing roles of double-stranded RNA-binding proteins in bacterial resistance.

The Arabidopsis plasma membrane-localized resistance protein RPM1 is degraded upon the induction of the hypersensitive response (HR) triggered in response to its own activation or that of other unrelated resistance (R) proteins. We investigated the role of RPM1 turnover in RPM1-mediated resistance and showed that degradation of RPM1 is not associated with HR or resistance mediated by this R protein. Likewise, the runaway cell death phenotype in the lsd1 mutant was not associated with RPM1 degradation and did not alter RPM1-derived resistance. RPM1 stability and RPM1-mediated resistance were dependent on the double-stranded RNA binding (DRB) proteins 1 and 4. Interestingly, the function of DRB1 in RPM1-mediated resistance was not associated with its role in pre-miRNA processing. The DRB3 and DRB5 proteins negatively regulated RPM1-mediated resistance and a mutation in these completely or partially restored resistance in the drb1, drb2, and drb4 mutant backgrounds. Conversely, plants overexpressing DRB5 showed attenuated RPM1-mediated resistance. A similar role for DRBs in basal and R-mediated resistance suggests that these proteins play a general role in bacterial resistance.

Signaling mechanisms underlying systemic acquired resistance to microbial pathogens.

Plants respond to biotic stress by inducing a variety of responses, which not only protect against the immediate diseases but also provide immunity from future infections. One example is systemic acquired resistance (SAR), which provides long-lasting and broad-spectrum protection at the whole plant level. The induction of SAR prepares the plant for a more robust response to subsequent infections from related and unrelated pathogens. SAR involves the rapid generation of signals at the primary site of infection, which are transported to the systemic parts of the plant presumably via the phloem. SAR signal generation and perception requires an intact cuticle, a waxy layer covering all aerial parts of the plant. A chemically diverse set of SAR inducers has already been identified, including hormones (salicylic acid, methyl salicylate), primary/secondary metabolites (nitric oxide, reactive oxygen species, glycerol-3-phosphate, azelaic acid, pipecolic acid, dihyroabetinal), fatty acid/lipid derivatives (18 carbon unsaturated fatty acids, galactolipids), and proteins (DIR1-Defective in Induced Resistance 1, AZI1-Azelaic acid Induced 1). Some of these are demonstrably mobile and the phloem loading routes for three of these SAR inducers is known. Here we discuss the recent findings related to synthesis, transport, and the relationship between these various SAR inducers.

Pipecolic acid confers systemic immunity by regulating free radicals.

Pipecolic acid (Pip), a non-proteinaceous product of lysine catabolism, is an important regulator of immunity in plants and humans alike. In plants, Pip accumulates upon pathogen infection and has been associated with systemic acquired resistance (SAR). However, the molecular mechanisms underlying Pip-mediated signaling and its relationship to other known SAR inducers remain unknown. We show that in plants, Pip confers SAR by increasing levels of the free radicals, nitric oxide (NO), and reactive oxygen species (ROS), which act upstream of glycerol-3-phosphate (G3P). Plants defective in NO, ROS, G3P, or salicylic acid (SA) biosynthesis accumulate reduced Pip in their distal uninfected tissues although they contain wild-type-like levels of Pip in their infected leaves. These data indicate that de novo synthesis of Pip in distal tissues is dependent on both SA and G3P and that distal levels of SA and G3P play an important role in SAR. These results also suggest a unique scenario whereby metabolites in a signaling cascade can stimulate each other's biosynthesis depending on their relative levels and their site of action.

Plants Pack a Quiver Full of Arrows.

Systemic acquired resistance (SAR) is a process wherein plants use chemical signals to communicate broad-spectrum systemic immunity to distant tissue. Two studies recently identified N-hydroxypipecolic acid as an additional essential SAR inducer. These findings assemble another piece in the SAR puzzle.

COP1, a negative regulator of photomorphogenesis, positively regulates plant disease resistance via double-stranded RNA binding proteins.

The E3 ubiquitin ligase COP1 (Constitutive Photomorphogenesis 1) is a well known component of the light-mediated plant development that acts as a repressor of photomorphogenesis. Here we show that COP1 positively regulates defense against turnip crinkle virus (TCV) and avrRPM1 bacteria by contributing to stability of resistance (R) protein HRT and RPM1, respectively. HRT and RPM1 levels and thereby pathogen resistance is significantly reduced in the cop1 mutant background. Notably, the levels of at least two double-stranded RNA binding (DRB) proteins DRB1 and DRB4 are reduced in the cop1 mutant background suggesting that COP1 affects HRT stability via its effect on the DRB proteins. Indeed, a mutation in either drb1 or drb4 resulted in degradation of HRT. In contrast to COP1, a multi-subunit E3 ligase encoded by anaphase-promoting complex (APC) 10 negatively regulates DRB4 and TCV resistance but had no effect on DRB1 levels. We propose that COP1-mediated positive regulation of HRT is dependent on a balance between COP1 and negative regulators that target DRB1 and DRB4.

Fatty Acid- and Lipid-Mediated Signaling in Plant Defense.

Fatty acids and lipids, which are major and essential constituents of all plant cells, not only provide structural integrity and energy for various metabolic processes but can also function as signal transduction mediators. Lipids and fatty acids can act as both intracellular and extracellular signals. In addition, cyclic and acyclic products generated during fatty acid metabolism can also function as important chemical signals. This review summarizes the biosynthesis of fatty acids and lipids and their involvement in pathogen defense.

Signaling Mechanisms Underlying Resistance Responses: What Have We Learned, and How Is It Being Applied?

Plants have evolved highly specific mechanisms to resist pathogens including preformed barriers and the induction of elaborate signaling pathways. Induced signaling requires recognition of the pathogen either via conserved pathogen-derived factors or specific pathogen-encoded proteins called effectors. Recognition of these factors by host encoded receptor proteins can result in the elicitation of different tiers of resistance at the site of pathogen infection. In addition, plants induce a type of systemic immunity which is effective at the whole plant level and protects against a broad spectrum of pathogens. Advances in our understanding of pathogen-recognition mechanisms, identification of the underlying molecular components, and their significant conservation across diverse plant species has enabled the development of novel strategies to combat plant diseases. This review discusses key advances in plant defense signaling that have been adapted or have the potential to be adapted for plant protection against microbial diseases.

Role of plasmodesmata and plasmodesmata localizing proteins in systemic immunity.

Systemic acquired resistance (SAR) is a highly desirable form of resistance that protects against a broad-spectrum of pathogens. SAR involves the generation of a mobile signal at the site of primary infection, which arms distal portions of a plant against subsequent secondary infections. A number of diverse chemical signals contributing to SAR have been isolated and characterized. Among these, salicylic acid (SA) functions in parallel to azelaic acid (AzA) and glycerol-3-phosphate (G3P), and both AzA and G3P function downstream of the free radicals nitric oxide and reactive oxygen species. We now show that phloem loading of AzA and G3P occurs via the symplast, whereas that of SA occurs via the apoplast. The symplastic transport of AzA and G3P is regulated by plasmodesmata localizing protein (PDLP) 5, which together with PDLP1 also plays a signaling role in SAR. Together, these results reveal the transport routes of SAR associated chemical signals, and the regulatory role of PDLPs in SAR.

The Potyviral P3 Protein Targets Eukaryotic Elongation Factor 1A to Promote the Unfolded Protein Response and Viral Pathogenesis.

The biochemical function of the potyviral P3 protein is not known, although it is known to regulate virus replication, movement, and pathogenesis. We show that P3, the putative virulence determinant of soybean mosaic virus (SMV), targets a component of the translation elongation complex in soybean. Eukaryotic elongation factor 1A (eEF1A), a well-known host factor in viral pathogenesis, is essential for SMV virulence and the associated unfolded protein response (UPR). Silencing GmEF1A inhibits accumulation of SMV and another ER-associated virus in soybean. Conversely, endoplasmic reticulum (ER) stress-inducing chemicals promote SMV accumulation in wild-type, but not GmEF1A-knockdown, plants. Knockdown of genes encoding the eEF1B isoform, which is important for eEF1A function in translation elongation, has similar effects on UPR and SMV resistance, suggesting a link to translation elongation. P3 and GmEF1A promote each other's nuclear localization, similar to the nuclear-cytoplasmic transport of eEF1A by the Human immunodeficiency virus 1 Nef protein. Our results suggest that P3 targets host elongation factors resulting in UPR, which in turn facilitates SMV replication and place eEF1A upstream of BiP in the ER stress response during pathogen infection.

Cooperative functioning between phenylalanine ammonia lyase and isochorismate synthase activities contributes to salicylic acid biosynthesis in soybean.

Salicylic acid (SA), an essential regulator of plant defense, is derived from chorismate via either the phenylalanine ammonia lyase (PAL) or the isochorismate synthase (ICS) catalyzed steps. The ICS pathway is thought to be the primary contributor of defense-related SA, at least in Arabidopsis. We investigated the relative contributions of PAL and ICS to defense-related SA accumulation in soybean (Glycine max). Soybean plants silenced for five PAL isoforms or two ICS isoforms were analyzed for SA concentrations and SA-derived defense responses to the hemibiotrophic pathogens Pseudomonas syringae and Phytophthora sojae. We show that, unlike in Arabidopsis, PAL and ICS pathways are equally important for pathogen-induced SA biosynthesis in soybean. Knock-down of either pathway shuts down SA biosynthesis and abrogates pathogen resistance. Moreover, unlike in Arabidopsis, pathogen infection is associated with the suppression of ICS gene expression. Pathogen-induced biosynthesis of SA via the PAL pathway correlates inversely with phenylalanine concentrations. Although infections with either virulent or avirulent strains of the pathogens increase SA concentrations, resistance protein-mediated response to avirulent P. sojae strains may function in an SA-independent manner. These results show that PAL- and ICS-catalyzed reactions function cooperatively in soybean defense and highlight the importance of PAL in pathogen-induced SA biosynthesis.