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.

Enhanced Disease Susceptibility1 Mediates Pathogen Resistance and Virulence Function of a Bacterial Effector in Soybean.

Enhanced disease susceptibility1 (EDS1) and phytoalexin deficient4 (PAD4) are well-known regulators of both basal and resistance (R) protein-mediated plant defense. We identified two EDS1-like (GmEDS1a/GmEDS1b) proteins and one PAD4-like (GmPAD4) protein that are required for resistance signaling in soybean (Glycine max). Consistent with their significant structural conservation to Arabidopsis (Arabidopsis thaliana) counterparts, constitutive expression of GmEDS1 or GmPAD4 complemented the pathogen resistance defects of Arabidopsis eds1 and pad4 mutants, respectively. Interestingly, however, the GmEDS1 and GmPAD4 did not complement pathogen-inducible salicylic acid accumulation in the eds1/pad4 mutants. Furthermore, the GmEDS1a/GmEDS1b proteins were unable to complement the turnip crinkle virus coat protein-mediated activation of the Arabidopsis R protein Hypersensitive reaction to Turnip crinkle virus (HRT), even though both interacted with HRT. Silencing GmEDS1a/GmEDS1b or GmPAD4 reduced basal and pathogen-inducible salicylic acid accumulation and enhanced soybean susceptibility to virulent pathogens. The GmEDS1a/GmEDS1b and GmPAD4 genes were also required for Resistance to Pseudomonas syringae pv glycinea2 (Rpg2)-mediated resistance to Pseudomonas syringae. Notably, the GmEDS1a/GmEDS1b proteins interacted with the cognate bacterial effector AvrA1 and were required for its virulence function in rpg2 plants. Together, these results show that despite significant structural similarities, conserved defense signaling components from diverse plants can differ in their functionalities. In addition, we demonstrate a role for GmEDS1 in regulating the virulence function of a bacterial effector.

Soybean NDR1-like proteins bind pathogen effectors and regulate resistance signaling.

Nonrace specific disease resistance 1 (NDR1) is a conserved downstream regulator of resistance (R) protein-derived signaling. We identified two NDR1-like sequences (GmNDR1a, b) from soybean, and investigated their roles in R-mediated resistance and pathogen effector detection. Silencing GmNDR1a and b in soybean shows that these genes are required for resistance derived from the Rpg1-b, Rpg3, and Rpg4 loci, against Pseudomonas syringae (Psg) expressing avrB, avrB2 and avrD1, respectively. Immunoprecipitation assays show that the GmNDR1 proteins interact with the AvrB2 and AvrD1 Psg effectors. This correlates with the enhanced virulence of Psg avrB2 and Psg avrD1 in GmNDR1-silenced rpg3 rpg4 plants, even though these strains are not normally more virulent on plants lacking cognate R loci. The GmNDR1 proteins interact with GmRIN4 proteins, but not with AvrB, or its cognate R protein Rpg1-b. However, the GmNDR1 proteins promote AvrB-independent activation of Rpg1-b when coexpressed with a phosphomimic derivative of GmRIN4b. The role of GmNDR1 proteins in Rpg1-b activation, their direct interactions with AvrB2/AvrD1, and a putative role in the virulence activities of Avr effectors, provides the first experimental evidence in support of the proposed role for NDR1 in transducing extracellular pathogen-derived signals.

Effect of stationary magnetic field strengths of 150 and 200 mT on reactive oxygen species production in soybean.

Our previous investigation reported the beneficial effect of pre-sowing magnetic treatment for improving germination parameters and biomass accumulation in soybean. In this study, soybean seeds treated with static magnetic fields of 150 and 200 mT for 1 h were evaluated for reactive oxygen species (ROS) and activity of antioxidant enzymes. Superoxide and hydroxyl radicals were measured in embryos and hypocotyls of germinating seeds by electron paramagnetic resonance spectroscopy and kinetics of superoxide production; hydrogen peroxide and antioxidant activities were estimated spectrophotometrically. Magnetic field treatment resulted in enhanced production of ROS mediated by cell wall peroxidase while ascorbic acid content, superoxide dismutase and ascorbate peroxidase activity decreased in the hypocotyl of germinating seeds. An increase in the cytosolic peroxidase activity indicated that this antioxidant enzyme had a vital role in scavenging the increased H(2)O(2) produced in seedlings from the magnetically treated seeds. Hence, these studies contribute to our first report on the biochemical basis of enhanced germination and seedling growth in magnetically treated seeds of soybean in relation to increased production of ROS.

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.

Enhancement of germination, growth, and photosynthesis in soybean by pre-treatment of seeds with magnetic field.

Experiments were conducted to study the effect of static magnetic fields on the seeds of soybean (Glycine max (L.) Merr. var: JS-335) by exposing the seeds to different magnetic field strengths from 0 to 300 mT in steps of 50 mT for 30, 60, and 90 min. Treatment with magnetic fields improved germination-related parameters like water uptake, speed of germination, seedling length, fresh weight, dry weight and vigor indices of soybean seeds under laboratory conditions. Improvement over untreated control was 5-42% for speed of germination, 4-73% for seedling length, 9-53% for fresh weight, 5-16% for dry weight, and 3-88% and 4-27% for vigor indices I and II, respectively. Treatment of 200 mT (60 min) and 150 mT (60 min), which were more effective than others in increasing most of the seedling parameters, were further explored for their effect on plant growth, leaf photosynthetic efficiency, and leaf protein content under field conditions. Among different growth parameters, leaf area, and leaf fresh weight showed maximum enhancement (more than twofold) in 1-month-old plants. Polyphasic chlorophyll a fluorescence (OJIP) transients from magnetically treated plants gave a higher fluorescence yield at the J-I-P phase. The total soluble protein map (SDS-polyacrylamide gel) of leaves showed increased intensities of the bands corresponding to a larger subunit (53 KDa) and smaller subunit (14 KDa) of Rubisco in the treated plants. We report here the beneficial effect of pre-sowing magnetic treatment for improving germination parameters and biomass accumulation in soybean.

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.

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.

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.

Plasmodesmata Localizing Proteins Regulate Transport and Signaling during Systemic Acquired Immunity in Plants.

Systemic acquired resistance (SAR) in plants is mediated by the signaling molecules azelaic acid (AzA), glycerol-3-phosphate (G3P), and salicylic acid (SA). Here, we show that AzA and G3P transport occurs via the symplastic route, which is regulated by channels known as plasmodesmata (PD). In contrast, SA moves via the extracytosolic apoplast compartment. We found that PD localizing proteins (PDLP) 1 and 5 were required for SAR even though PD permeability in pdlp1 and 5 mutants was comparable to or higher than wild-type plants, respectively. Furthermore, PDLP function was required in the recipient cell, suggesting regulatory function in SAR. Interestingly, overexpression of PDLP5 drastically reduced PD permeability, yet also impaired SAR. PDLP1 interacted with AZI1 (lipid transfer-like protein required for AzA- and G3P-induced SAR) and contributed to its intracellular partitioning. Together, these results reveal the transport routes of SAR chemical signals and highlight the regulatory role of PD-localizing proteins in SAR.

Nitric oxide and reactive oxygen species are required for systemic acquired resistance in plants.

Systemic acquired resistance (SAR) is a form of broad-spectrum disease resistance that is induced in response to primary infection and that protects uninfected portions of the plant against secondary infections by related or unrelated pathogens. SAR is associated with an increase in chemical signals that operate in a collective manner to confer protection against secondary infections. These include, the phytohormone salicylic acid (SA), glycerol-3-phosphate (G3P), azelaic acid (AzA) and more recently identified signals nitric oxide (NO) and reactive oxygen species (ROS). NO, ROS, AzA and G3P function in the same branch of the SAR pathway, and in parallel to the SA-regulated branch. NO and ROS function upstream of AzA/G3P and different reactive oxygen species functions in an additive manner to mediate chemical cleavage of the C9 double bond on C18 unsaturated fatty acids to generate AzA. The parallel and additive functioning of various chemical signals provides important new insights in the overlapping pathways leading to SAR.

Mono- and digalactosyldiacylglycerol lipids function nonredundantly to regulate systemic acquired resistance in plants.

The plant galactolipids monogalactosyldiacylglycerol (MGDG) and digalactosyldiacylglycerol (DGDG) have been linked to the anti-inflammatory and cancer benefits of a green leafy vegetable diet in humans due to their ability to regulate the levels of free radicals like nitric oxide (NO). Here, we show that DGDG contributes to plant NO as well as salicylic acid biosynthesis and is required for the induction of systemic acquired resistance (SAR). In contrast, MGDG regulates the biosynthesis of the SAR signals azelaic acid (AzA) and glycerol-3-phosphate (G3P) that function downstream of NO. Interestingly, DGDG is also required for AzA-induced SAR, but MGDG is not. Notably, transgenic expression of a bacterial glucosyltransferase is unable to restore SAR in dgd1 plants even though it does rescue their morphological and fatty acid phenotypes. These results suggest that MGDG and DGDG are required at distinct steps and function exclusively in their individual roles during the induction of SAR.

Free radicals mediate systemic acquired resistance.

Systemic acquired resistance (SAR) is a form of resistance that protects plants against a broad spectrum of secondary infections. However, exploiting SAR for the protection of agriculturally important plants warrants a thorough investigation of the mutual interrelationships among the various signals that mediate SAR. Here, we show that nitric oxide (NO) and reactive oxygen species (ROS) serve as inducers of SAR in a concentration-dependent manner. Thus, genetic mutations that either inhibit NO/ROS production or increase NO accumulation (e.g., a mutation in S-nitrosoglutathione reductase [GSNOR]) abrogate SAR. Different ROS function additively to generate the fatty-acid-derived azelaic acid (AzA), which in turn induces production of the SAR inducer glycerol-3-phosphate (G3P). Notably, this NO/ROS→AzA→G3P-induced signaling functions in parallel with salicylic acid-derived signaling. We propose that the parallel operation of NO/ROS and SA pathways facilitates coordinated regulation in order to ensure optimal induction of SAR.

Oxyradicals and PSII activity in maize leaves in the absence of UV components of solar spectrum.

The regulation of oxyradicals and PSII activity by UV-B (280-315 nm) and UV-A (315-400 nm) components were investigated in the leaves of maize [Zea mays L. var: HQPM.1]. The impact of ambient UV radiation on the production of superoxide (O(2)(·-)) and hydroxyl ((·)OH) radicals were analysed in the leaves of 20-day-old plants. The amount of O(2)(·-) and (·)OH radicals and the radical scavenging activity were significantly higher in the leaves exposed to ambient UV radiation as compared to the leaves of the plants grown under UV exclusion filters. Smaller amount of oxyradicals in the leaves of UV excluded plants was accompanied by a substantial increase in quantum yield of electron transport (φ(Eo)), rate of electron transport (ψ(o)) and performance index (PI(ABS)), as indicated by chlorophyll a fluorescence transient. Although higher amounts of oxyradicals invoked higher activity of antioxidant enzymes like superoxide dismutase and peroxidase under ambient UV, they also imposed limitation on the photosynthetic efficiency of PSII. Exclusion of UV components (UV-B 280-315 nm; UV-A 315-400 nm) translated to enhanced photosynthesis, growth and biomass. Thus, solar UV components, especially in the tropical region, could be a major limiting factor in the photosynthetic efficiency of the crop plants.