Direct Detection of Tobacco Mosaic Virus in Infected Plants with SERS-Sensing Hydrogels.

The impact of plant pathogens on global crop yields is a major societal concern. The current agricultural diagnostic paradigm involves either visual inspection (inaccurate) or laboratory molecular tests (burdensome). While field-ready diagnostic methods have advanced in recent years, issues remain with detection of presymptomatic infections, multiplexed analysis, and requirement for in-field sample processing. To overcome these issues, we developed surface-enhanced Raman scattering (SERS)-sensing hydrogels that detect pathogens through simple contact with a leaf. In this work, we developed a novel reagentless SERS sensor for the detection of tobacco mosaic virus (TMV) and embedded it in an optimized hydrogel material to produce sensing hydrogels. To test the diagnostic application of our sensing hydrogels, we demonstrate their use to detect TMV infection in tobacco plants. This technology has the potential to shift the current agricultural diagnostic paradigm by offering a field-deployable tool for presymptomatic and multiplexed molecular identification of pathogens.

Rapid and Reliable Quantification of Glycerol-3-phosphate Using Gas Chromatography-coupled Mass Spectrometry.

Glycerol-3-phosphate (G3P) is a conserved precursor of glycerolipids that also plays an important role in plant defense. Its levels and/or metabolism are also associated with many human disorders including insulin resistance, diabetes, obesity, and cancer, among others. In plants, G3P accumulates upon pathogen infection and is a critical component of systemic acquired resistance, which confers broad spectrum disease resistance against secondary infections. G3P also plays an important role in root-shoot-root signaling in soybean that regulates incompatible interactions with nitrogen-fixing bacteria. Thus, accurate quantification of G3P is key to drawing a valid conclusion regarding its role in diverse processes ranging from lipid biosynthesis to defense. G3P quantification is further compounded by its rapid degradation in extracts prepared at room temperature. Here, we describe a simplified procedure for accurate quantitative analysis of G3P from plant tissues. G3P was extracted along with the internal standard ribitol, derivatized with N-Methyl-N-(trimethylsilyl)trifluoroacetamide (MSTFA) and analyzed by gas chromatography-coupled mass spectrometry using selective ion mode. This procedure is simple, economical, and efficient, and does not involve isotopic internal standards or multiple-step derivatizations.

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.

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.

An Emerging Role for Chloroplasts in Disease and Defense.

Chloroplasts are key players in plant immune signaling, contributing to not only de novo synthesis of defensive phytohormones but also the generation of reactive oxygen and nitrogen species following activation of pattern recognition receptors or resistance (R) proteins. The local hypersensitive response (HR) elicited by R proteins is underpinned by chloroplast-generated reactive oxygen species. HR-induced lipid peroxidation generates important chloroplast-derived signaling lipids essential to the establishment of systemic immunity. As a consequence of this pivotal role in immunity, pathogens deploy effector complements that directly or indirectly target chloroplasts to attenuate chloroplast immunity (CI). Our review summarizes the current knowledge of CI signaling and highlights common pathogen chloroplast targets and virulence strategies. We address emerging insights into chloroplast retrograde signaling in immune responses and gaps in our knowledge, including the importance of understanding chloroplast heterogeneity and chloroplast involvement in intraorganellular interactions in host immunity.

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.

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.

Pipecolic Acid Quantification Using Gas Chromatography-coupled Mass Spectrometry.

Pipecolic acid (Pip), a non-proteinacious product of lysine catabolism, is an important regulator of immunity in plants and humans alike. For instance, Pip accumulation is associated with the genetic disorder Zellweger syndrome, chronic liver diseases, and pyridoxine-dependent epilepsy in humans. In plants, Pip accumulates upon pathogen infection and is required for plant defense. The aminotransferase ALD1 catalyzes biosynthesis of Pip precursor piperideine-2-carboxylic acid, which is converted to Pip via ornithine cyclodeaminase. A variety of methods are used to quantify Pip, and some of these involve use of expensive amino acid analysis kits. Here, we describe a simplified procedure for quantitative analysis of Pip from plant tissues. Pipecolic acid was extracted from leaf tissues along with an internal standard norvaline, derivatized with propyl chloroformate and analyzed by gas chromatography-coupled mass spectrometry using selective ion mode. This procedure is simple, economical, and efficient and does not involve isotopic internal standards or multiple-step derivatizations.

JMJ14 encoded H3K4 demethylase modulates immune responses by regulating defence gene expression and pipecolic acid levels.

Epigenetic modifications have emerged as an important mechanism underlying plant defence against pathogens. We examined the role of JMJ14, a Jumonji (JMJ) domain-containing H3K4 demethylase, in local and systemic plant immune responses in Arabidopsis. The function of JMJ14 in local or systemic defence response was investigated by pathogen growth assays and by analysing expression and H3K4me3 enrichments of key defence genes using qPCR and ChIP-qPCR. Salicylic acid (SA) and pipecolic acid (Pip) levels were quantified and function of JMJ14 in SA- and Pip-mediated defences was analysed in Col-0 and jmj14 plants. jmj14 mutants were compromised in both local and systemic defences. JMJ14 positively regulates pathogen-induced H3K4me3 enrichment and expression of defence genes involved in SA- and Pip-mediated defence pathways. Consequently, loss of JMJ14 results in attenuated defence gene expression and reduced Pip accumulation during establishment of systemic acquired resistance (SAR). Exogenous Pip partially restored SAR in jmj14 plants, suggesting that JMJ14 regulated Pip biosynthesis and other downstream factors regulate SAR in jmj14 plants. JMJ14 positively modulates defence gene expressions and Pip levels in Arabidopsis.

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.