Genome-Wide Identification of the NPR1-like Gene Family in Solanum tuberosum and Functional Characterization of StNPR1 in Resistance to Ralstonia solanacearum.

The NPR1 (nonexpressor of pathogenesis-related genes 1) gene is an activator of the systemic acquisition of resistance (SAR) in plants and is one of the central factors in their response to pathogenic bacterial infestation, playing an important role in plant disease resistance. Potato (Solanum tuberosum) is a crucial non-grain crop that has been extensively studied. However, the identification and analysis of the NPR1-like gene within potato have not been understood well. In this study, a total of six NPR1-like proteins were identified in potato, and phylogenetic analysis showed that the six NPR1-like proteins in Solanum tuberosum could be divided into three major groups with NPR1-related proteins from Arabidopsis thaliana and other plants. Analysis of the exon-intron patterns and protein domains of the six NPR1-like genes from potato showed that the exon-intron patterns and protein domains of the NPR1-like genes belonging to the same Arabidopsis thaliana subfamily were similar. By performing quantitative real-time PCR (qRT-PCR) analysis, we found that six NPR1-like proteins have different expression patterns in different potato tissues. In addition, the expression of three StNPR1 genes was significantly downregulated after being infected by Ralstonia solanacearum (RS), while the difference in the expression of StNPR2/3 was insignificant. We also established potato StNPR1 overexpression lines that showed a significantly increased resistance to R. solanacearum and elevated activities of chitinase, ß-1,3-glucanase, and phenylalanine deaminase. Increased peroxidase (POD), superoxide dismutase (SOD), and catalase (CAT) activities, as well as decreased hydrogen peroxide, regulated the dynamic balance of reactive oxygen species (ROS) in the StNPR1 overexpression lines. The transgenic plants activated the expression of the genes associated with the Salicylic acid (SA) defense response but suppressed the expression of the genes associated with Jasmonic acid (JA) signaling. This resulted in resistance to Ralstonia solanacearum.

The WAK-like protein RFO1 acts as a sensor of the pectin methylation status in Arabidopsis cell walls to modulate root growth and defense.

Most organisms adjust their development according to the environmental conditions. For the majority, this implies the sensing of alterations to cell walls caused by different cues. Despite the relevance of this process, few molecular players involved in cell wall sensing are known and characterized. Here, we show that the wall-associated kinase-like protein RESISTANCE TO FUSARIUM OXYSPORUM 1 (RFO1) is required for plant growth and early defense against Fusarium oxysporum and functions by sensing changes in the pectin methylation levels in the cell wall. The RFO1 dwell time at the plasma membrane is affected by the pectin methylation status at the cell wall, regulating MITOGEN-ACTIVATED PROTEIN KINASE and gene expression. We show that the extracellular domain of RFO1 binds de-methylated pectin in vitro, whose distribution in the cell wall is altered during F. oxysporum infection. Further analyses also indicate that RFO1 is required for the BR-dependent plant growth alteration in response to inhibition of pectin de-methyl-esterase activity at the cell wall. Collectively, our work demonstrates that RFO1 is a sensor of the pectin methylation status that plays a unique dual role in plant growth and defense against vascular pathogens.

Functional Genetic Diversity and Plant Growth Promoting Potential of Polyphosphate Accumulating Bacteria in Soil.

Polyphosphate (polyP) accumulation is an important trait of microorganisms. Implication of polyP accumulating bacteria (PAB) in enhanced biological phosphate removal, heavy metal sequestration, and dissolution of dental enamel is well studied. Phosphorous (P) accumulated within microbial biomass also regulates labile P in soil; however, abundance and diversity of the PAB in soil is still unexplored. Present study investigated the genetic and functional diversity of PAB in rhizosphere soil. Here, we report the abundance of Pseudomonas spp. as high PAB in soil, suggesting their contribution to global P cycling. Additional subset analysis of functional genes i.e., polyphosphate kinase (ppk) and exopolyphosphatase (ppx) in all PAB, indicates their significance in bacterial growth and metabolism. Distribution of functional genes in phylogenetic tree represent a more biologically realistic discrimination for the two genes. Distribution of ppx gene disclosed its phylogenetic conservation at species level, however, clustering of ppk gene of similar species in different clades illustrated its environmental condition mediated modifications. Selected PAB showed tolerance to abiotic stress and strong correlation with plant growth promotary (PGP) traits viz. phosphate solubilization, auxin and siderophore production. Interaction of PAB with A. thaliana enhanced the growth and phosphate status of the plant under salinity stress, suggestive of their importance in P cycling and stress alleviation. IMPORTANCE Study discovered the abundance of Pseudomonas genera as a high phosphate accumulator in soil. The presence of functional genes (polyphosphate kinase [ppk] and exopolyphosphatase [ppx]) in all PAB depicts their importance in polyphosphate metabolism in bacteria. Genetic and functional diversity reveals conservation of the ppx gene at species level. Furthermore, we found a positive correlation between PAB and plant growth promotary traits, stress tolerance, and salinity stress alleviation in A. thaliana.

Extracellular DNA as an elicitor of broad-spectrum resistance in Arabidopsis thaliana.

Like in mammals, the plant immune system has evolved to perceive damage. Damaged-associated molecular patterns (DAMPs) are endogenous signals generated in wounded or infected tissue after pathogen or insect attack. Although extracellular DNA (eDNA) is a DAMP signal that induces immune responses, plant responses after eDNA perception remain largely unknown. Here, we report that signaling defenses but not direct defense responses are induced after eDNA applications enhancing broad-range plant protection. A screening of defense signaling and hormone biosynthesis marker genes revealed that OXI1, CML37 and MPK3 are relevant eDNA-Induced Resistance markers (eDNA-IR). Additionally, we observed that eDNA from several Arabidopsis ecotypes and other phylogenetically distant plants such as citrus, bean and, more surprisingly, a monocotyledonous plant such as maize upregulates eDNA-IR marker genes. Using 3,3'-Diaminobenzidine (DAB) and aniline blue staining methods, we observed that H2O2 but not callose was strongly accumulated following self-eDNA treatments. Finally, eDNA resulted in effective induced resistance in Arabidopsis against the pathogens Hyaloperonospora arabidopsidis, Pseudomonas syringae, and Botrytis cinerea and against aphid infestation, reducing the number of nymphs and moving forms. Hence, the unspecificity of DNA origin and the wide range of insects to which eDNA can protect opens many questions about the mechanisms behind eDNA-IR.

GhMYB4 downregulates lignin biosynthesis and enhances cotton resistance to Verticillium dahliae.

KEY MESSAGE: GhMYB4 acts as a negative regulator in lignin biosynthesis, which results in alteration of cell wall integrity and activation of cotton defense response. Verticillium wilt of cotton (Gossypium hirsutum) caused by the soil-borne fungus Verticillium dahliae (V. dahliae) represents one of the most important constraints of cotton production worldwide. Mining of the genes involved in disease resistance and illuminating the molecular mechanisms that underlie this resistance is of great importance in cotton breeding programs. Defense-induced lignification in plants is necessary for innate immunity, and there are reports of a correlation between increased lignification and disease resistance. In this study, we present an example in cotton whereby plants with reduced lignin content also exhibit enhanced disease resistance. We identified a negative regulator of lignin synthesis, in cotton encoded in GhMYB4. Overexpression of GhMYB4 in cotton and Arabidopsis enhanced resistance to V. dahliae  with reduced lignin deposition. Moreover, GhMYB4 could bind the promoters of several genes involved in lignin synthesis, such as GhC4H-1, GhC4H-2, Gh4CL-4, and GhCAD-3, and impair their expression. The reduction of lignin content in GhMYB4-overexpressing cotton led to alterations of cell wall integrity (CWI) and released more oligogalacturonides (OGs) which may act as damage-associated molecular patterns (DAMPs) to stimulate plant defense responses. In support of this hypothesis, exogenous application with polygalacturonic acid (PGA) in cotton activated biosynthesis of jasmonic acid (JA) and JA-mediated defense against V. dahliae, similar to that described for cotton plants overexpressing GhMYB4. This study provides a new candidate gene for cotton disease-resistant breeding and an increased understanding of the relationship between lignin synthesis, OG release, and plant immunity.

AtPME17 is a functional Arabidopsis thaliana pectin methylesterase regulated by its PRO region that triggers PME activity in the resistance to Botrytis cinerea.

Pectin is synthesized in a highly methylesterified form in the Golgi cisternae and partially de-methylesterified in muro by pectin methylesterases (PMEs). Arabidopsis thaliana produces a local and strong induction of PME activity during the infection of the necrotrophic fungus Botrytis cinerea. AtPME17 is a putative A. thaliana PME highly induced in response to B. cinerea. Here, a fine tuning of AtPME17 expression by different defence hormones was identified. Our genetic evidence demonstrates that AtPME17 strongly contributes to the pathogen-induced PME activity and resistance against B. cinerea by triggering jasmonic acid-ethylene-dependent PDF1.2 expression. AtPME17 belongs to group 2 isoforms of PMEs characterized by a PME domain preceded by an N-terminal PRO region. However, the biochemical evidence for AtPME17 as a functional PME is still lacking and the role played by its PRO region is not known. Using the Pichia pastoris expression system, we demonstrate that AtPME17 is a functional PME with activity favoured by an increase in pH. AtPME17 performs a blockwise pattern of pectin de-methylesterification that favours the formation of egg-box structures between homogalacturonans. Recombinant AtPME17 expression in Escherichia coli reveals that the PRO region acts as an intramolecular inhibitor of AtPME17 activity.

Metabolic and Genomic Traits of Phytobeneficial Phenazine-Producing Pseudomonas spp. Are Linked to Rhizosphere Colonization in Arabidopsis thaliana and Solanum tuberosum.

Bacterial rhizosphere colonization is critical for phytobeneficial rhizobacteria such as phenazine-producing Pseudomonas spp. To better understand this colonization process, potential metabolic and genomic determinants required for rhizosphere colonization were identified using a collection of 60 phenazine-producing Pseudomonas strains isolated from multiple plant species and representative of the worldwide diversity. Arabidopsis thaliana and Solanum tuberosum (potato) were used as host plants. Bacterial rhizosphere colonization was measured by quantitative PCR using a newly designed primer pair and TaqMan probe targeting a conserved region of the phenazine biosynthetic operon. The metabolic abilities of the strains were assessed on 758 substrates using Biolog phenotype microarray technology. These data, along with available genomic sequences for all strains, were analyzed in light of rhizosphere colonization. Strains belonging to the P. chlororaphis subgroup colonized the rhizospheres of both plants more efficiently than strains belonging to the P. fluorescens subgroup. Metabolic results indicated that the ability to use amines and amino acids was associated with an increase in rhizosphere colonization capability in A. thaliana and/or in S. tuberosum The presence of multiple genetic determinants in the genomes of the different strains involved in catabolic pathways and plant-microbe and microbe-microbe interactions correlated with increased or decreased rhizosphere colonization capabilities in both plants. These results suggest that the metabolic and genomic traits found in different phenazine-producing Pseudomonas strains reflect their rhizosphere competence in A. thaliana and S. tuberosum Interestingly, most of these traits are associated with similar rhizosphere colonizing capabilities in both plant species.IMPORTANCE Rhizosphere colonization is crucial for plant growth promotion and biocontrol by antibiotic-producing Pseudomonas spp. This colonization process relies on different bacterial determinants which partly remain to be uncovered. In this study, we combined a metabolic and a genomic approach to decipher new rhizosphere colonization determinants which could improve our understanding of this process in Pseudomonas spp. Using 60 distinct strains of phenazine-producing Pseudomonas spp., we show that rhizosphere colonization abilities correlated with both metabolic and genomic traits when these bacteria were inoculated on two distant plants, Arabidopsis thaliana and Solanum tuberosum Key metabolic and genomic determinants presumably required for efficient colonization of both plant species were identified. Upon further validation, these targets could lead to the development of simple screening tests to rapidly identify efficient rhizosphere colonizers.

The effects of soil phosphorus content on plant microbiota are driven by the plant phosphate starvation response.

Phosphate starvation response (PSR) in nonmycorrhizal plants comprises transcriptional reprogramming resulting in severe physiological changes to the roots and shoots and repression of plant immunity. Thus, plant-colonizing microorganisms-the plant microbiota-are exposed to direct influence by the soil's phosphorus (P) content itself as well as to the indirect effects of soil P on the microbial niches shaped by the plant. The individual contribution of these factors to plant microbiota assembly remains unknown. To disentangle these direct and indirect effects, we planted PSR-deficient Arabidopsis mutants in a long-term managed soil P gradient and compared the composition of their shoot and root microbiota to wild-type plants across different P concentrations. PSR-deficiency had a larger effect on the composition of both bacterial and fungal plant-associated microbiota than soil P concentrations in both roots and shoots. To dissect plant-microbe interactions under variable P conditions, we conducted a microbiota reconstitution experiment. Using a 185-member bacterial synthetic community (SynCom) across a wide P concentration gradient in an agar matrix, we demonstrated a shift in the effect of bacteria on the plant from a neutral or positive interaction to a negative one, as measured by rosette size. This phenotypic shift was accompanied by changes in microbiota composition: the genus Burkholderia was specifically enriched in plant tissue under P starvation. Through a community drop-out experiment, we demonstrated that in the absence of Burkholderia from the SynCom, plant shoots accumulated higher ortophosphate (Pi) levels than shoots colonized with the full SynCom but only under Pi starvation conditions. Therefore, Pi-stressed plants are susceptible to colonization by latent opportunistic competitors found within their microbiome, thus exacerbating the plant's Pi starvation.

PMR5, an acetylation protein at the intersection of pectin biosynthesis and defense against fungal pathogens.

Powdery mildew (Golovinomyces cichoracearum), one of the most prolific obligate biotrophic fungal pathogens worldwide, infects its host by penetrating the plant cell wall without activating the plant's innate immune system. The Arabidopsis mutant powdery mildew resistant 5 (pmr5) carries a mutation in a putative pectin acetyltransferase gene that confers enhanced resistance to powdery mildew. Here, we show that heterologously expressed PMR5 protein transfers acetyl groups from [14 C]-acetyl-CoA to oligogalacturonides. Through site-directed mutagenesis, we show that three amino acids within a highly conserved esterase domain in putative PMR5 orthologs are necessary for PMR5 function. A suppressor screen of mutagenized pmr5 seed selecting for increased powdery mildew susceptibility identified two previously characterized genes affecting the acetylation of plant cell wall polysaccharides, RWA2 and TBR. The rwa2 and tbr mutants also suppress powdery mildew disease resistance in pmr6, a mutant defective in a putative pectate lyase gene. Cell wall analysis of pmr5 and pmr6, and their rwa2 and tbr suppressor mutants, demonstrates minor shifts in cellulose and pectin composition. In direct contrast to their increased powdery mildew resistance, both pmr5 and pmr6 plants are highly susceptibile to multiple strains of the generalist necrotroph Botrytis cinerea, and have decreased camalexin production upon infection with B. cinerea. These results illustrate that cell wall composition is intimately connected to fungal disease resistance and outline a potential route for engineering powdery mildew resistance into susceptible crop species.

The nematicide Serratia plymuthica M24T3 colonizes Arabidopsis thaliana, stimulates plant growth, and presents plant beneficial potential.

Nine bacterial strains were previously isolated in association with pinewood nematode (PWN) from wilted pine trees. They proved to be nematicidal in vitro, and one of the highest activities, with potential to control PWN, was showed by Serratia sp. M24T3. Its ecology in association with plants remains unclear. This study aimed to evaluate the ability of strain M24T3 to colonize the internal tissues of the model plant Arabidopsis thaliana using confocal microscopy. Plant growth-promoting bacteria (PGPB) functional traits were tested and retrieved in the genome of strain M24T3. In greenhouse conditions, the bacterial effects of all nematicidal strains were also evaluated, co-inoculated or not with Bradyrhizobium sp. 3267, on Vigna unguiculata fitness. Inoculation of strain M24T3 increased the number of A. thaliana lateral roots and the confocal analysis confirmed effective bacterial colonization in the plant. Strain M24T3 showed cellulolytic activity, siderophores production, phosphate and zinc solubilization ability, and indole acetic acid production independent of supplementation with L-tryptophan. In the genome of strain M24T3, genes involved in the interaction with the plants such as 1-aminocyclopropane-1-carboxylate (ACC) deaminase, chitinolytic activity, and quorum sensing were also detected. The genomic organization showed ACC deaminase and its leucine-responsive transcriptional regulator, and the activity of ACC deaminase was 594.6 nmol α-ketobutyrate µg protein-1 µl-1. Strain M24T3 in co-inoculation with Bradyrhizobium sp. 3267 promoted the growth of V. unguiculata. In conclusion, this study demonstrated the ability of strain M24T3 to colonize other plants besides pine trees as an endophyte and displays PGPB traits that probably increased plant tolerance to stresses.

Plant-mediated effects of soil phosphorus on the root-associated fungal microbiota in Arabidopsis thaliana.

Plants respond to phosphorus (P) limitation through an array of morphological, physiological and metabolic changes which are part of the phosphate (Pi) starvation response (PSR). This response influences the establishment of the arbuscular mycorrhizal (AM) symbiosis in most land plants. It is, however, unknown to what extent available P and the PSR redefine plant interactions with the fungal microbiota in soil. Using amplicon sequencing of the fungal taxonomic marker ITS2, we examined the changes in root-associated fungal communities in the AM nonhost species Arabidopsis thaliana in response to soil amendment with P and to genetic perturbations in the plant PSR. We observed robust shifts in root-associated fungal communities of P-replete plants in comparison with their P-deprived counterparts, while bulk soil communities remained unaltered. Moreover, plants carrying mutations in the phosphate signaling network genes, phr1, phl1 and pho2, exhibited similarly altered root fungal communities characterized by the depletion of the chytridiomycete taxon Olpidium brassicae specifically under P-replete conditions. This study highlights the nutritional status and the underlying nutrient signaling network of an AM nonhost plant as previously unrecognized factors influencing the assembly of the plant fungal microbiota in response to P in nonsterile soil.

TBL10 is required for O-acetylation of pectic rhamnogalacturonan-I in Arabidopsis thaliana.

O-Acetylated pectins are abundant in the primary cell wall of plants and growing evidence suggests they have important roles in plant cell growth and interaction with the environment. Despite their importance, genes required for O-acetylation of pectins are still largely unknown. In this study, we showed that TRICHOME BIREFRINGENCE LIKE 10 (AT3G06080) is involved in O-acetylation of pectins in Arabidopsis (Arabidopsis thaliana). The activity of the TBL10 promoter was strong in tissues where pectins are highly abundant (e.g. leaves). Two homozygous knock-out mutants of Arabidopsis, tbl10-1 and tbl10-2, were isolated and shown to exhibit reduced levels of wall-bound acetyl esters, equivalent of ~50% of the wild-type level in pectin-enriched fractions derived from leaves. Further fractionation revealed that the degree of acetylation of the pectin rhamnogalacturonan-I (RG-I) was reduced in the tbl10 mutant compared to the wild type, whereas the pectin homogalacturonan (HG) was unaffected. The degrees of acetylation in hemicelluloses (i.e. xyloglucan, xylan and mannan) were indistinguishable between the tbl10 mutants and the wild type. The mutant plants contained normal trichomes in leaves and exhibited a similar level of susceptibility to the phytopathogenic microorganisms Pseudomonas syringae pv. tomato DC3000 and Botrytis cinerea; while they displayed enhanced tolerance to drought. These results indicate that TBL10 is required for O-acetylation of RG-I, possibly as an acetyltransferase, and suggest that O-acetylated RG-I plays a role in abiotic stress responses in Arabidopsis.

Biofilm formation and regulation of salicylic acid-inducible genes expression in Arabidopsis by Algerian indigenous bacteria from wheat and potatoes rhizospheres in semi-arid Sétif region.

45 bacterial isolates from potatoes and wheat rhizospheres near Sétif (Algeria) pre-selected for their antagonistic activity against three fungal plant pathogens, two necrotrophic Fusarium solani var. coeruleum and Phytophtora infestans, and a systemic F. oxysporum f. sp. albedinis. Molecular typing of the isolates showed abundance of Bacillus compared to Pseudomonas. Some of the tested strains have shown very high biofilm formation. Among the 24 Gram-positive bacilli screened for four cyclic lipopeptides genes, some isolates harbor two or more genes, while others have a single gene or have none. Four selected isolates were able to regulate the expression of six defense-related genes in Arabidopsis and produce salicylic acid. Upon the features assessed in this study, strain B. amyloliquefaciens A16 was selected for a subsequent use as seed treatment and biocontrol agent in semi-arid region fields. This strain showed important biofilm formation, regulation of Arabidopsis defenses, and harbored three cLPs genes.

A Comparison of the Antibacterial and Antifungal Activities of Thiosulfinate Analogues of Allicin.

Allicin (diallylthiosulfinate) is a defence molecule from garlic (Allium sativum L.) with broad antimicrobial activities in the low µM range against Gram-positive and -negative bacteria, including antibiotic resistant strains, and fungi. Allicin reacts with thiol groups and can inactivate essential enzymes. However, allicin is unstable at room temperature and antimicrobial activity is lost within minutes upon heating to >80 °C. Allicin's antimicrobial activity is due to the thiosulfinate group, so we synthesized a series of allicin analogues and tested their antimicrobial properties and thermal stability. Dimethyl-, diethyl-, diallyl-, dipropyl- and dibenzyl-thiosulfinates were synthesized and tested in vitro against bacteria and the model fungus Saccharomyces cerevisiae, human and plant cells in culture and Arabidopsis root growth. The more volatile compounds showed significant antimicrobial properties via the gas phase. A chemogenetic screen with selected yeast mutants showed that the mode of action of the analogues was similar to that of allicin and that the glutathione pool and glutathione metabolism were of central importance for resistance against them. Thiosulfinates differed in their effectivity against specific organisms and some were thermally more stable than allicin. These analogues could be suitable for applications in medicine and agriculture either singly or in combination with other antimicrobials.

Alginate-perlite encapsulated Pseudomonas putida A (ATCC 12633) cells: Preparation, characterization and potential use as plant inoculants.

Microbial immobilization can be used to prepare encapsulated inoculants. Here, we characterize and describe the preparation of Ca-alginate-perlite microbeads loaded with cells of plant growth-promoting Pseudomonas putida A (ATCC 12633), for their future application as agricultural inoculants. The microbeads were prepared by dropwise addition of a CaCl2-paraffin emulsion mixture to an emulsion containing alginate 2% (w/v), perlite 0.1-0.4% (w/v) and bacterial suspension in 0.9% NaCl (1010 CFU/mL). For all perlite concentrations used, microbead size was 90-120 µm, the trapped population was 108 CFU/g microbeads and the increase in mechanical stability was proportional to perlite concentration. Microbeads containing 0.4% (w/v) perlite were able to release bacteria into the medium after 30 days of incubation. When we evaluated how P. putida A (ATCC 12633) entrapped in Ca-alginate-perlite (0.4% (w/v)) microbeads colonized the Arabidopsis thaliana rhizosphere, an increase in colonization over time was detected (from an initial 2.1 × 104 to 9.2 × 105 CFU/g soil after 21 days). With this treatment, growth promotion of A. thaliana occurred with an increase in the amount of proteins, and in root and leaf biomass. It was concluded that the microbeads could be applied as possible inoculants, since they provide protection and a controlled release of microorganisms into the rhizosphere.

A conserved RxLR effector interacts with host RABA-type GTPases to inhibit vesicle-mediated secretion of antimicrobial proteins.

Plant pathogens of the oomycete genus Phytophthora produce virulence factors, known as RxLR effector proteins that are transferred into host cells to suppress disease resistance. Here, we analyse the function of the highly conserved RxLR24 effector of Phytophthora brassicae. RxLR24 was expressed early in the interaction with Arabidopsis plants and ectopic expression in the host enhanced leaf colonization and zoosporangia formation. Co-immunoprecipitation (Co-IP) experiments followed by mass spectrometry identified different members of the RABA GTPase family as putative RxLR24 targets. Physical interaction of RxLR24 or its homologue from the potato pathogen Phytophthora infestans with different RABA GTPases of Arabidopsis or potato, respectively, was confirmed by reciprocal Co-IP. In line with the function of RABA GTPases in vesicular secretion, RxLR24 co-localized with RABA1a to vesicles and the plasma membrane. The effect of RxLR24 on the secretory process was analysed with fusion constructs of secreted antimicrobial proteins with a pH-sensitive GFP tag. PATHOGENESIS RELATED PROTEIN 1 (PR-1) and DEFENSIN (PDF1.2) were efficiently exported in control tissue, whereas in the presence of RxLR24 they both accumulated in the endoplasmic reticulum. Together our results imply a virulence function of RxLR24 effectors as inhibitors of RABA GTPase-mediated vesicular secretion of antimicrobial PR-1, PDF1.2 and possibly other defence-related compounds.

A Pectin Methylesterase Inhibitor Enhances Resistance to Verticillium Wilt.

Pectins are major components of the primary plant cell wall, which functions as the primary barrier against pathogens. Pectin methylesterases (PMEs) catalyze the demethylesterification of the homogalacturonan domains of pectin in the plant cell wall. Their activity is regulated by PME inhibitors (PMEIs). Here, we provide evidence that the pectin methylesterase-inhibiting protein GhPMEI3 from cotton (Gossypium hirsutum) functions in plant responses to infection by the fungus Verticillium dahliae GhPMEI3 interacts with PMEs and regulates the expression of a specific fungal polygalacturonase (VdPG1). Ectopic expression of GhPMEI3 increased pectin methyl esterification and limited fungal disease in cotton, while also modulating root elongation. Enzymatic analyses revealed that GhPMEI3 efficiently inhibited the activity of cotton GhPME2/GhPME31. Experiments using transgenic Arabidopsis (Arabidopsis thaliana) plants expressing the GhPMEI3 gene under the control of the CaMV 35S promoter revealed that GhPMEI3 inhibits the endogenous PME activity in vitro. Moreover, the enhanced resistance to V. dahliae was associated with altered VdPG1 expression. Virus-induced silencing of GhPMEI3 resulted in increased susceptibility to V. dahliae Further, we investigated the interaction between GhPMEI3 and GhPME2/GhPME31 using inhibition assays and molecular docking simulations. The peculiar structural features of GhPMEI3 were responsible for the formation of a 1:1 stoichiometric complex with GhPME2/GhPME31. Together, these results suggest that GhPMEI3 enhances resistance to Verticillium wilt. Moreover, GhPMEI3-GhPMEs interactions would be needed before drawing the correlation between structure-function and are crucial for plant development against the ever-evolving fungal pathogens.

Systemic changes in photosynthesis and reactive oxygen species homeostasis in shoots of Arabidopsis thaliana infected with the beet cyst nematode Heterodera schachtii.

Photosynthetic efficiency and redox homeostasis are important for plant physiological processes during regular development as well as defence responses. The second-stage juveniles of Heterodera schachtii induce syncytial feeding sites in host roots. To ascertain whether the development of syncytia alters photosynthesis and the metabolism of reactive oxygen species (ROS), chlorophyll a fluorescence measurements and antioxidant responses were studied in Arabidopsis thaliana shoots on the day of inoculation and at 3, 7 and 15 days post-inoculation (dpi). Nematode parasitism caused an accumulation of superoxide and hydrogen peroxide molecules in the shoots of infected plants at 3 dpi, probably as a result of the observed down-regulation of antioxidant enzymes. These changes were accompanied by an increase in RNA and lipid oxidation markers. The activities of antioxidant enzymes were found to be enhanced on infection at 7 and 15 dpi, and the content of anthocyanins was elevated from 3 dpi. The fluorescence parameter Rfd , defining plant vitality and the photosynthetic capacity of leaves, decreased by 11% only at 7 dpi, and non-photochemical quenching (NPQ), indicating the effectiveness of photoprotection mechanisms, was about 16% lower at 3 and 7 dpi. As a result of infection, the ultrastructure of chloroplasts was changed (large starch grains and plastoglobules), and more numerous and larger peroxisomes were observed in the mesophyll cells of leaves. We postulate that the joint action of antioxidant enzymes/molecules and photochemical mechanisms leading to the maintenance of photosynthetic efficiency promotes the fine-tuning of the infected plants to oxidative stress induced by parasitic cyst nematodes.

Genomewide identification of genes involved in the potato response to drought indicates functional evolutionary conservation with Arabidopsis plants.

Potato is one of the four most important food crop plants worldwide and is strongly affected by drought. The following two pairs of potato cultivars, which are related in ancestry but show different drought tolerances, were chosen for comparative gene expression studies: Gwiazda/Oberon and Tajfun/Owacja. Comparative RNA-seq analyses of gene expression differences in the transcriptomes obtained from drought-tolerant versus drought-sensitive plants during water shortage conditions were performed. The 23 top-ranking genes were selected, 22 of which are described here as novel potato drought-responsive genes. Moreover, all but one of the potato genes selected have homologues in the Arabidopsis genome. Of the seven tested A. thaliana mutants with altered expression of the selected homologous genes, compared to the wild-type Arabidopsis plants, six showed an improved tolerance to drought. These genes encode carbohydrate transporter, mitogen-activated protein kinase kinase kinase 15 (MAPKKK15), serine carboxypeptidase-like 19 protein (SCPL19), armadillo/beta-catenin-like repeat-containing protein, high-affinity nitrate transporter 2.7 and nonspecific lipid transfer protein type 2 (nsLPT). The evolutionary conservation of the functions of the selected genes in the plant response to drought confirms the importance of these identified potato genes in the ability of plants to cope with water shortage conditions. Knowledge regarding these gene functions can be used to generate potato cultivars that are resistant to unfavourable conditions. The approach used in this work and the obtained results allowed for the identification of new players in the plant response to drought.

Piriformospora indica promotes early flowering in Arabidopsis through regulation of the photoperiod and gibberellin pathways.

Flowering in plants is synchronized by both environmental cues and internal regulatory factors. Previous studies have shown that the endophytic fungus Piriformospora indica promotes the growth and early flowering in Coleus forskohlii (a medicinal plant) and Arabidopsis. To further dissect the impact of P. indica on pathways responsible for flowering time in Arabidopsis, we co-cultivated Arabidopsis with P. indica and used RT-qPCR to analyze the main gene regulation networks involved in flowering. Our results revealed that the symbiotic interaction of Arabidopsis with P. indica promotes early flower development and the number of siliques. In addition, expression of the core flowering regulatory gene FLOWERING LOCUS T (FT), of genes controlling the photoperiod [CRYPTOCHROMES (CRY1, CRY2) and PHYTOCHROME B (PHYB)] and those related to gibberellin (GA) functions (RGA1, AGL24, GA3, and MYB5) were induced by the fungus, while key genes controlling the age and autonomous pathways remained unchanged. Moreover, early flowering promotion conferred by P. indica was promoted by exogenous GA and inhabited by GA inhibitor, and this effect could be observed under long day and neutral day photoperiod. Therefore, our data suggested that P. indica promotes early flowering in Arabidopsis likely through photoperiod and GA rather than age or the autonomous pathway.