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Chemical fungicides have been instrumental in protecting crops from fungal diseases. However, mounting fungal resistance to many of the single-site chemical fungicides calls for the development of new antifungal agents with novel modes of action (MoA). The sequence-divergent cysteine-rich antifungal defensins with multi-site MoA are promising starting templates for design of novel peptide-based fungicides. Here, we experimentally tested such a set of 17-amino acid peptides containing the γ-core motif of the antifungal plant defensin MtDef4. These designed peptides exhibited antifungal properties different from those of MtDef4. Focused analysis of a lead peptide, GMA4CG_V6, showed it was a random coil in solution with little or no secondary structure elements. Additionally, it exhibited potent cation-tolerant antifungal activity against the plant fungal pathogenBotrytis cinerea, causal agent of gray mold disease in fruits and vegetables. Its multi-site MoA involved localization predominantly to the plasma membrane, permeabilization of the plasma membrane, rapid internalization into the vacuole and cytoplasm, and affinity for bioactive phosphoinositides phosphatidylinositol 3-phosphate (PI3P), PI4P, and PI5P. The sequence motif RRRW was identified as a major determinant of the antifungal activity of this peptide. While topical spray-application of GMA4CG_V6 onNicotiana benthamianaand tomato plants provided preventative and curative suppression of gray mold disease symptoms, the peptide was not internalized into plant cells. Our findings open the possibility that truncated and modified defensin-derived peptides containing the γ-core sequence could serve as promising candidates for further development as bioinspired fungicides.

The auxin indole-3-acetic acid (IAA) is a plant hormone that not only regulates plant growth and development but also plays important roles in plant-microbe interactions. We previously reported that IAA alters expression of several virulence-related genes in the plant pathogen Pseudomonas syringae pv. tomato strain DC3000 (PtoDC3000). To learn more about the impact of IAA on regulation of PtoDC3000 gene expression we performed a global transcriptomic analysis of bacteria grown in culture, in the presence or absence of exogenous IAA. We observed that IAA repressed expression of genes involved in the Type III secretion (T3S) system and motility and promoted expression of several known and putative transcriptional regulators. Several of these regulators are orthologs of factors known to regulate stress responses and accordingly expression of several stress response-related genes was also upregulated by IAA. Similar trends in expression for several genes were also observed by RT-qPCR. Using an Arabidopsis thaliana auxin receptor mutant that accumulates elevated auxin, we found that many of the P. syringae genes regulated by IAA in vitro were also regulated by auxin in planta. Collectively the data indicate that IAA modulates many aspects of PtoDC3000 biology, presumably to promote both virulence and survival under stressful conditions, including those encountered in or on plant leaves. IMPORTANCE Indole-3-acetic acid (IAA), a form of the plant hormone auxin, is used by many plant-associated bacteria as a cue to sense the plant environment. Previously, we showed that IAA can promote disease in interactions between the plant pathogen Pseudomonas syringae strain PtoDC000 and one of its hosts, Arabidopsis thaliana. However, the mechanisms by which IAA impacts the biology of PtoDC3000 and promotes disease are not well understood. Here we demonstrate that IAA is a signal molecule that regulates gene expression in PtoDC3000. The presence of exogenous IAA affects expression of over 700 genes in the bacteria, including genes involved in Type III secretion and genes involved in stress response. This work offers insight into the roles of auxin promoting pathogenesis.

The auxin indole-3-acetic acid (IAA) is a plant hormone that not only regulates plant growth and development but also plays important roles in plant-microbe interactions. We previously reported that IAA alters expression of several virulence-related genes in the plant pathogen Pseudomonas syringae pv. tomato strain DC3000 (PtoDC3000). To learn more about the impact of IAA on regulation of PtoDC3000 gene expression, we performed a global transcriptomic analysis of bacteria grown in culture, in the presence or absence of exogenous IAA. We observed that IAA repressed expression of genes involved in the type III secretion (T3S) system and motility and promoted expression of several known and putative transcriptional regulators. Several of these regulators are orthologs of factors known to regulate stress responses and accordingly expression of several stress response-related genes was also upregulated by IAA. Similar trends in expression for several genes were also observed by quantitative reverse transcription PCR. Using an Arabidopsis thaliana auxin receptor mutant that accumulates elevated auxin, we found that many of the P. syringae genes regulated by IAA in vitro were also regulated by auxin in planta. Collectively the data indicate that IAA modulates many aspects of PtoDC3000 biology, presumably to promote both virulence and survival under stressful conditions, including those encountered in or on plant leaves. IMPORTANCE Indole-3-acetic acid (IAA), a form of the plant hormone auxin, is used by many plant-associated bacteria as a cue to sense the plant environment. Previously, we showed that IAA can promote disease in interactions between the plant pathogen Pseudomonas syringae strain PtoDC000 and one of its hosts, Arabidopsis thaliana. However, the mechanisms by which IAA impacts the biology of PtoDC3000 and promotes disease are not well understood. Here, we demonstrate that IAA is a signal molecule that regulates gene expression in PtoDC3000. The presence of exogenous IAA affects expression of over 700 genes in the bacteria, including genes involved in type III secretion and genes involved in stress response. This work offers insight into the roles of auxin-promoting pathogenesis.

microRNAs regulate dynamic aspects of innate immunity in Arabidopsis thaliana in response to lipopolysaccharides. Lectin-domain receptor-like kinases function as surveillance proteins and miR393 targets transcripts of an L-type LecRK (LECRK-V.7, At3g59740). This study investigated miR393 regulation of LecRLKs associated with LPS perception. Following pre-treatment of wild type -, miR393 ab double mutant - and miR393 overexpressor plants with LPS, the expression of miR393 and two other LecRLK genes (G-type lectin S-receptor-like protein kinases, SD1-13 (At1g11330) and SD1-29 (At1g61380) were evaluated. Overexpression and repression of miR393 respectively suppressed and induced transcripts of the LecRLK genes. The results indicate that miR393 regulates the three LecRLKs following perception of bacterial LPS, in support of immunity and basal resistance.

Modification of host hormone biology is a common strategy used by plant pathogens to promote disease. For example, the bacterial pathogen strain Pseudomonas syringae DC3000 (PtoDC3000) produces the plant hormone auxin (indole-3-acetic acid [IAA]) to promote PtoDC3000 growth in plant tissue. Previous studies suggest that auxin may promote PtoDC3000 pathogenesis through multiple mechanisms, including both suppression of salicylic acid (SA)-mediated host defenses and via an unknown mechanism that appears to be independent of SA. To test if host auxin signaling is important during pathogenesis, we took advantage of Arabidopsis thaliana lines impaired in either auxin signaling or perception. We found that disruption of auxin signaling in plants expressing an inducible dominant axr2–1 mutation resulted in decreased bacterial growth and that this phenotype was suppressed by introducing the sid2–2 mutation, which impairs SA synthesis. Thus, host auxin signaling is required for normal susceptibility to PtoDC3000 and is involved in suppressing SA-mediated defenses. Unexpectedly, tir1 afb1 afb4 afb5 quadruple-mutant plants lacking four of the six known auxin coreceptors that exhibit decreased auxin perception, supported increased levels of bacterial growth. This mutant exhibited elevated IAA levels and reduced SA-mediated defenses, providing additional evidence that auxin promotes disease by suppressing host defense. We also investigated the hypothesis that IAA promotes PtoDC3000 virulence through a direct effect on the pathogen and found that IAA modulates expression of virulence genes, both in culture and in planta. Thus, in addition to suppressing host defenses, IAA acts as a microbial signaling molecule that regulates bacterial virulence gene expression.

Modification of host hormone biology is a common strategy used by plant pathogens to promote disease. For example, the bacterial pathogenPseudomonas syringaestrainPtoDC3000 produces the plant hormone auxin (Indole-3-acetic acid, or IAA) to promotePtoDC3000 growth in plant tissue. Previous studies suggest that auxin may promotePtoDC3000 pathogenesis through multiple mechanisms, including both suppression of salicylic acid (SA)-mediated host defenses and via an unknown mechanism that appears to be independent of SA. To test if host auxin signaling is important during pathogenesis, we took advantage ofArabidopsis thalianalines impaired in either auxin signaling or perception. We found that disruption of auxin signaling in plants expressing an inducible dominantaxr2-1mutation resulted in decreased bacterial growth, demonstrating that host auxin signaling is required for normal susceptibility toPtoDC3000, and this phenotype was dependent on SA-mediated defenses. However, despite exhibiting decreased auxin perception,tir1 afb1 afb4 afb5quadruple mutant plants lacking four of the six known auxin co-receptors supported increased levels of bacterial growth. This mutant also exhibited elevated IAA levels, suggesting that the increased IAA in these plants may promotePtoDC3000 growth independent of host auxin signaling, perhaps through a direct effect on the pathogen. In support of this, we found that IAA directly impacted the pathogen, by modulating expression of bacterial virulence genes, both in liquid culture and in planta. Thus, in addition to suppressing host defenses, IAA acts as a microbial signaling molecule that regulates bacterial virulence gene expression.

Defense responses of tomato (Solanum lycopersicum L.) against attack by Pectobacterium carotovorum subsp. carotovorum (Pcc), the causal agent of soft rot diseases, were studied. The expression of some tomato defense genes were evaluated by real‐time PCR quantification analysis, 24 and 72 hr after actively growing tomato plants were inoculated with Pcc. These included: MYB transcriptor factor, ethylene response element‐binding protein, suppressor of the G2 allele of Skp1, cytochrome P450, small Sar1 GTPase, hydroxycinnamoyl‐CoA:quinate hydroxycinnamoyl transferase, pathogenesis‐related protein 1a, endo‐1,3‐beta‐glucanase, chitinase, proteinase inhibitor, defensin, CC‐NBS‐LRR resistance protein, and phenylalanine ammonia lyase. The results showed dynamic transcriptomic changes, with transcripts exhibiting different expression kinetics at 24 and 72 hr to confer resistance to tomato against Pcc infection., Targeting various disease response‐related genes, real‐time PCR was performed on tomato leaf tissue sampled 24 and 72 hr postinoculation with Pectobacterium carotovorum subsp. carotovorum (Pcc). The results showed dynamic transcriptomic changes, with transcripts exhibiting different expression kinetics at 24 and 72 hr to confer resistance to tomato against Pcc infection.

The metabolome of a biological system provides a functional readout of the cellular state, thus serving as direct signatures of biochemical events that define the dynamic equilibrium of metabolism and the correlated phenotype. Hence, to elucidate biochemical processes involved in sorghum responses to fungal infection, a liquid chromatography-mass spectrometry-based untargeted metabolomic study was designed. Metabolic alterations of three sorghum cultivars responding to Colletotrichum sublineolum, were investigated. At the 4-leaf growth stage, the plants were inoculated with fungal spore suspensions and the infection monitored over time: 0, 3, 5, 7, and 9 days post inoculation. Non-infected plants were used as negative controls. The metabolite composition of aqueous-methanol extracts were analyzed on an ultra-high performance liquid chromatography system coupled to high-definition mass spectrometry. The acquired multidimensional data were processed to create data matrices for multivariate statistical analysis and chemometric modeling. The computed chemometric models indicated time- and cultivar-related metabolic changes that reflect sorghum responses to the fungal infection. Metabolic pathway and correlation-based network analyses revealed that this multi-component defense response is characterized by a functional metabolic web, containing defense-related molecular cues to counterattack the pathogen invasion. Components of this network are metabolites from a range of interconnected metabolic pathways with the phenylpropanoid and flavonoid pathways being the central hub of the web. One of the key features of this altered metabolism was the accumulation of an array of phenolic compounds, particularly de novo biosynthesis of the antifungal 3-deoxyanthocynidin phytoalexins, apigeninidin, luteolinidin, and related conjugates. The metabolic results were complemented by qRT-PCR gene expression analyses that showed upregulation of defense-related marker genes. Unraveling key characteristics of the biochemical mechanism underlying sorghum-C. sublineolum interactions, provided valuable insights with potential applications in breeding crop plants with enhanced disease resistance. Furthermore, the study contributes to ongoing efforts toward a comprehensive understanding of the regulation and reprogramming of plant metabolism under biotic stress.

The metabolome of a biological system provides a functional readout of the cellular state, thus serving as direct signatures of biochemical events that define the dynamic equilibrium of metabolism and the correlated phenotype. Hence, to elucidate biochemical processes involved in sorghum responses to fungal infection, a liquid chromatography-mass spectrometry-based untargeted metabolomic study was designed. Metabolic alterations of three sorghum cultivars responding to

Although aortic wave reflection may be inherited, the extent to which indexes of wave reflection derived from wave separation analysis (reflected (backward) wave index (RI) and pressure (Pb)) show intrafamilial aggregation and heritability is uncertain. We therefore aimed to determine the intrafamilial aggregation and heritability of RI and Pb and compare these with indexes of pressure augmentation.Aortic Pb, RI, augmented pressure (Pa), and augmentation index (AIx) were determined using radial applanation tonometry and SphygmoCor software in 1,152 participants of 315 families (111 father-mother, 705 parent-child, and 301 sibling-sibling pairs) from an urban developing community of black Africans. Heritability estimates were determined from Statistical Analysis for Genetic Epidemiology software.With appropriate adjustments, significant correlations were noted between parent-child pairs for Pb and Pa (P0.05 for all), but not for RI (P = 0.50) or AIx (P = 0.90) and between sib-sib pairs for Pb and Pa (P0.05), but not for RI (P = 0.54) or AIx (P = 0.14). No correlations for indexes of wave reflection were noted between fathers and mothers (P0.57). After adjustments, Pb (h2 = 0.24±0.07) and Pa (h2 = 0.23±0.07) (P0.001 for both) but not RI (h2 = 0.04±0.06, P = 0.27) or AIx (h2 = 0.10±0.07, P = 0.07) showed significant heritability.Aortic reflected (backward) waves derived from either wave separation (Pb) or pulse wave analysis (Pa) show a similar degree of intrafamilial aggregation and heritability, but the use of RI or AIx may underestimate reflected wave effects.

Background Plants respond to various stress stimuli by activating an enhanced broad-spectrum defensive ability. The development of novel resistance inducers represents an attractive, alternative crop protection strategy. In this regard, hexanoic acid (Hxa, a chemical elicitor) and azelaic acid (Aza, a natural signaling compound) have been proposed as inducers of plant defense, by means of a priming mechanism. Here, we investigated both the mode of action and the complementarity of Aza and Hxa as priming agents in Nicotiana tabacum cells in support of enhanced defense. Results Metabolomic analyses identified signatory biomarkers involved in the establishment of a pre-conditioned state following Aza and Hxa treatment. Both inducers affected the metabolomes in a similar manner and generated common biomarkers: caffeoylputrescine glycoside, cis-5-caffeoylquinic acid, feruloylglycoside, feruloyl-3-methoxytyramine glycoside and feruloyl-3-methoxytyramine conjugate. Subsequently, quantitative real time-PCR was used to investigate the expression of inducible defense response genes: phenylalanine ammonia lyase, hydroxycinnamoyl CoA quinate transferase and hydroxycinnamoyl transferase to monitor activation of the early phenylpropanoid pathway and chlorogenic acids metabolism, while ethylene response element-binding protein, small sar1 GTPase, heat shock protein 90, RAR1, SGT1, non-expressor of PR genes 1 and thioredoxin were analyzed to report on signal transduction events. Pathogenesis-related protein 1a and defensin were quantified to investigate the activation of defenses regulated by salicylic acid and jasmonic acid respectively. The qPCR results revealed differential expression kinetics and, in general (except for NPR1, Thionin and PR1a), the relative gene expression ratios observed in the Hxa-treated cells were significantly greater than the expression observed in the cells treated with Aza. Conclusions The results indicate that Aza and Hxa have a similar priming effect through activation of genes involved in the establishment of systemic acquired resistance, associated with enhanced synthesis of hydroxycinnamic acids and related conjugates. Electronic supplementary material The online version of this article (10.1186/s12870-017-1157-5) contains supplementary material, which is available to authorized users.

Background Pectobacterium carotovorum subsp. carotovorum (Pcc) is a soft rot bacterium which upon entry into the plant macerates plant tissues by producing plant cell wall degrading enzymes. It has a wide host range which includes carrot, potato, tomato, leafy greens, squash and other cucurbits, onion, green peppers and cassava. During plant-microbe interactions, one of the ways of plant response to pathogen infection is through the small RNA silencing mechanism. Under pathogen attack the plant utilizes microRNAs to regulate gene expression by means of mediating gene silencing at transcriptional and post-transcriptional level. This study aims to assess for the first time, the expression profile of some stress-responsive miRNA and differential expression pattern of their target genes in Arabidopsis thaliana inoculated with Pcc. Materials and methods Leaves of five weeks old Arabidopsis thaliana plants were infected with Pcc and the quantitative real time-PCR, was used to investigate after 0, 24, 48 and 72 h post infection, the expression profiling of the stress-responsive miRNAs which include: miR156, miR159, miR169, miR393, miR396 miR398, miR399 and miR408 along with their target genes which include: Squamosa promoter-binding-like protein, myb domain protein 101, nuclear factor Y subunit A8, concanavalin A-like lectin protein kinase, growth regulating factor 4, copper superoxide dismutase, ubiquitin-protein ligase and plantacyanin respectively. Results The findings showed that the overexpression of 6 miRNAs at 24, 48 and 72 h after infection resulted in the repression of their target genes and the expression of 2 miRNAs didn't affect their target genes. Conclusion These results provide the first indication of the miRNAs role in response to the infection of Pcc in A. thaliana and open new vistas for a better understanding of miRNA regulation of plant response to Pcc.

MicroRNAs (miRNAs) are a class of small non-coding RNAs that have recently emerged as important regulators of gene expression, mainly through cleavage and/or translation inhibition of the target mRNAs during or after transcription. miRNAs play important roles by regulating a multitude of biological processes in plants which include maintenance of genome integrity, development, metabolism, and adaptive responses towards environmental stresses. The increasing population of the world and their food demands requires focused efforts for the improvement of crop plants to ensure sustainable food production. Manipulation of mRNA transcript abundance via miRNA control provides a unique strategy for modulating differential plant gene expression and miRNAs are thus emerging as the next generation targets for genetic engineering for improvement of the agronomic properties of crops. However, a deeper understanding of its potential and the mechanisms involved will facilitate the design of suitable strategies to obtain the desirable traits with minimum trade-offs in the modified crops. In this regard, this review highlights the diverse roles of conserved and newly identified miRNAs in various food and industrial crops and recent advances made in the uses of miRNAs to improve plants of agronomically importance so as to significantly enhance crop yields and increase tolerance to various environmental stress agents of biotic - or abiotic origin.

Background The extensive and intensive uses of organophosphorus insecticide—quinalphos in agriculture, pose a health hazard to animals, humans, and environment because of its persistence in the soil and crops. However, there is no much information available on the biodegradation of quinalphos by the soil micro-organisms, which play a significant role in detoxifying pesticides in the environment; so research is initiated in biodegradation of quinalphos. Results A soil bacterium strain, capable of utilizing quinalphos as its sole source of carbon and energy, was isolated from soil via the enrichment method on minimal salts medium (MSM). On the basis of morphological, biochemical and 16S rRNA gene sequence analysis, the bacterium was identified as to be Bacillus thuringiensis. Bacillus thuringiensis grew on quinalphos with a generation time of 28.38 min or 0.473 h in logarithmic phase. Maximum degradation of quinalphos was observed with an inoculum of 1.0 OD, an optimum pH (6.5–7.5), and an optimum temperature of 35–37 °C. Among the additional carbon and nitrogen sources, the carbon source—sodium acetate and nitrogen source—a yeast extract marginally improved the rate of degradation of quinalphos. Conclusions Display of degradation of quinalphos by B. thuringiensis in liquid culture in the present study indicates the potential of the culture for decontamination of quinalphos in polluted environment sites.

The anthracnose pathogen, Colletotrichum gloeosporioides (Penz.) Penz. & Sacc., is a major cause of disease in the avocado industry, causing significant economic losses, and infects all cultivars. In South Africa, cvs Fuerte and Hass are the most widely grown. Identification of genes differentially expressed in avocado during infection with the fungus represents an important step towards understanding the plants defence responses and would assist in designing appropriate intervention strategies. In this study, 454 sequencing and analysis of the transcriptome of infected cv. Fuerte avocado fruits were performed using the Roche 454 GS FLX Titanium platform. cDNA libraries enriched for differentially expressed genes were constructed from unharvested and harvested avocado fruit tissues collected after 1, 4 and 24 h postinfection (early response) and after 3, 4, 5 and 7 days postinfection (late response), then sequenced. RT-PCR was used to validate the sequencing results. The single sequencing run produced 215 781 reads from the transcriptome with an average sequence length of 252–300 nucleotides. A total of 70.6 megabases of sequence data were generated and subjected to BLAST searches from which 639 genes encoding proteins functioning in metabolism, signal transduction, transcriptional control, defence, stress, transportation processes and some genes with unknown functions were identified. Avocado is able to respond to C. gloeosporioides infection by exhibiting a sophisticated molecular system for pathogen recognition and by activating structural and biochemical defence mechanisms. This study represents the first transcriptome analysis of avocado fruit following infection with C. gloeosporioides, and the findings are discussed in relation to the known or putative functions of the gene products.

Chilli pepper (Capsicum annuum L.) is susceptible to Pectobacterium carotovorum subsp. carotovorum (Pcc), the causal agent of soft rot disease in crops. Understanding the molecular principles of systemic acquired resistance, which is poorly understood in chilli pepper, represents an important step towards understanding inducible defence responses and can assist in designing appropriate intervention strategies for crop disease management. Accordingly, we investigated (via real-time PCR and metabolomics profiling) the molecular response of chilli pepper to Pcc by characterisation of the crucial metabolic regulators involved in the establishment of defence response. We profiled 13 key inducible defence response genes, which included MYB transcriptor factor, ethylene response element-binding protein, suppressor of the G2 allele of Skp1, cytochrome P450, small Sar1 (GTPase), hydroxycinnamoyl-CoA:quinate hydroxycinnamoyl transferase, pathogenesis-related protein 1a, endo-1,3-β-glucanase, chitinase, proteinase inhibitor, defensin, coiled-coil-nucleotide-binding site-leucine-rich repeat (CC–NBS–LRR) resistance and phenylalanine ammonia lyase. In addition, we determined metabolomic shifts induced by Pcc in pepper. The PCR results revealed a significant induction of the selected plant defence-related genes in response to Pcc inoculation; the metabolomic profiling showed that of 99 primary metabolites profiled the quantities of acetylcarnitine, adenosine, adenosine 3′,5′ cyclic monophosphate, guanosine 3′,5′ cyclic monophosphate and inosine decreased in pepper leaves inoculated with Pcc.

An inability to show consistent relationships between gene variants and blood pressure (BP) may be confounded by the use of office BP measurement. Whether the difference between office BP and day BP (office-day) is genetically predetermined is unknown. We therefore aimed to determine the intrafamilial aggregation and heritability of office-day BP.Nurse-derived office BP (mean of 5 measurements according to guidelines) and 24-h ambulatory BP were determined for 592 participants from 198 families (67 spouse pairs, 361 parent-child pairs, and 169 sibling-sibling pairs), with 12 families having three generations, from an urban developing community of black Africans. Heritability estimates were determined using SAGE software.With adjustments for confounders, office systolic BP (SBP) (h=0.35±0.09, P0.0001) showed comparable heritability estimates to 24-h SBP (h=0.33±0.09, P0.0001). Similarly, with adjustments for confounders, office diastolic BP (DBP) (h=0.37±0.09, P0.0001) showed comparable heritability estimates as 24-h DBP (h=0.35±0.09, P0.0001). However, multivariate adjusted heritability estimates of day SBP (h=0.29±0.09, P0.0001) and DBP (h=0.33±0.09, P0.0001) were not diminished by further adjustments for office SBP (h=0.42±0.09, P0.0001) or DBP (h=0.34±0.09, P0.0001). Further, independent of confounders, office-day BP showed significant intrafamilial aggregation and heritability (SBP: h=0.51±0.10, P0.0001; DBP: h=0.37±0.09, P0.0001), effects that persisted with further adjustments for office, day, or day-night BP (P0.0005 for SBP and DBP).Although office and ambulatory BP may show similar heritability estimates, genetic associations with carefully determined office BP measurements may be confounded by the heritability of office-day BP differences.