Polygalacturonase-inhibiting Protein Research Articles

Overexpression of OsPGIP2 confers Sclerotinia sclerotiorum resistance in Brassica napus through increased activation of defense mechanisms.

Sclerotinia stem rot (SSR), caused by Sclerotinia sclerotiorum, is the most serious disease affecting the yield of the agriculturally and economically important crop Brassica napus (rapeseed). In this study, Oryza sativa polygalacturonase-inhibiting protein 2 (OsPGIP2) was found to effectively enhanced rapeseed immunity against S. sclerotiorum infection. Leaf extracts of B. napus plants overexpressing OsPGIP2 showed enhanced S. sclerotiorum resistance by delaying pathogen infection. The constitutive expression of OsPGIP2 in rapeseed plants provided a rapid and effective defense response, which included the production of reactive oxygen species, interactions with S. sclerotiorum polygalacturonases (SsPG3 and SsPG6), and effects on the expression of defense genes. RNA sequencing analysis revealed that the pathogen induced many differentially expressed genes associated with pathogen recognition, redox homeostasis, mitogen-activated protein kinase signaling cascades, hormone signaling pathways, pathogen-/defense-related genes, and cell wall-related genes. The overexpression of OsPGIP2 also led to constitutively increased cell wall cellulose and hemicellulose contents in stems without compromising seed quality. The results demonstrate that OsPGIP2 plays a major role in rapeseed defense mechanisms, and we propose a model for OsPGIP2-conferred resistance to S. sclerotiorum in these plants.

Lack of efficacy of transgenic pea (Pisum sativum L.) stably expressing antifungal genes against Fusarium spp. in three years of confined field trials

ABSTRACTFusarium root rot is a major pea disease in Canada and only partial tolerance exists in germplasm. Transgenic technologies may hold promise but the economic benefits of genetically modified (GM) pea will need to surpass the regulatory costs, time and labor involved in bringing a GM crop to market. European pea (Pisum sativum L.) cultivars expressing four antifungal genes, 1-3 β glucanase (G), endochitinase (C) (belonging to PR proteins family), polygalacturonase inhibiting proteins (PGIPs) (P) and stilbene synthase (V) have been transformed for disease tolerance and showed disease tolerance under laboratory conditions. Transgenic lines with four antifungal genes inserted either individually or stacked through crossing were tested for their efficacy against Fusarium root rot (Fusarium avenaceum) in confined trials over three years (2013 to 2015) in comparison with two parental German lines and three Canadian lines. Superior emergence, higher fresh weight or lower disease ratings above and below ground, of transgenic lines in presence of disease inoculum were not observed consistently in the three years of field experiments when compared to the parental and Canadian lines in the presence of disease inoculum. No indication of an advantage of stacked genes over single genes was observed. Most transgenic lines had lower relative gene expression in the roots than in the leaves in greenhouse trials suggesting a possible explanation for poor tolerance to Fusarium root rot. Field trials are necessary to verify the agronomic performance and ecological relevance of the promising effects detected under laboratory conditions.

Sugar beet polygalacturonase-inhibiting proteins with 11 LRRs confer Rhizoctonia, Fusarium and Botrytis resistance in Nicotiana plants

Polygalacturonase-inhibiting proteins (PGIPs) are leucine-rich repeat (LRR) proteins that inhibit polygalacturonase (PG) enzymes secreted by pathogens to break down plant cell walls during early stage of disease development. Sugar beet (Beta vulgaris L.) PGIP genes (BvPGIPs) have 11 LRR domains as compared to 10 LRRs generally found in other plant species. To determine whether the BvPGIPs have a function in plant defense, genes encoding BvPGIP1 and BvPGIP2 that differ in 8 amino acids were fused with the CaMV 35S constitutive promoter and introduced into Nicotiana benthamiana. Crude PGIP protein extracts from BvPGIP1 transgenic plants significantly inhibited Rhizoctonia solani, Fusarium solani and Botrytis cinerea PGs. BvPGIP2 extract also inhibited PGs from F. solani and B. cinerea but did not inhibit PGs from R. solani. When transgenic BvPGIP plants were bioassayed for resistance, similar results were obtained. This is a first report that documents sugar beet PGIPs with 11 LRRs confer resistance to three different fungal pathogens.

Exogenous potassium phosphite application improved PR-protein expression and associated physio-biochemical events in cucumber challenged by Pseudoperonospora cubensis

Abstract In the present study, the effect of potassium phosphite on Pseudoperonospora cubensis-inoculated cucumber plants was investigated. Different defense-related enzymes including laccase, polyphenoloxidase and glucanase as well as total protein and lignin contents were analyzed. Anatomical alterations in plant tissues were analyzed using a light microscope. Expression changes in major pathogenesis-related genes were studied at different time courses. The highest expression of glucanase was observed in pre-inoculated plants (97% higher than control) at 24 h with 97% increase compared to the control plants, while Chitinase transcripts were accumulated at a maximum level in potassium phosphite-treated plants 96 h after inoculation with 93% increase over control plants. Analysis of polygalacturonase inhibitor proteins gene expression revealed a transcription peak (96% increase over control plants) 48 h after inoculation. The potassium phosphite-treated plants exhibited an increase in β-1,3-glucanase (82%) enzymatic activity as well as total protein (53%), polyphenoloxidase (21%), laccase (11%) and lignin contents (15%) in comparison to the control. The results of the anatomical assay showed an increase in the vascular bundle diameter in potassium phosphite-treated plants (174 μm) and a decrease in pathogen-treated leaves (66 μm) compared with the control (100 μm). It can be suggested that potassium phosphite treatment induced higher expression of plant defense genes and increased laccase and polyphenoloxidase activities which in turn enhanced lignin deposition in plant tissues. The findings of the present study would be implemented for designing a controlling program to decrease the adverse effect of Pseudoperonospora cubensis on cucumber plants.

Molecular cloning and functional analysis of two novel polygalacturonase genes in Rhizoctonia solani

Two novel polygalacturonase (PG) genes, RsPG3 and RsPG4, were cloned from Rhizoctonia solani isolate YN-7, which is a member of anastomosis group (AG) subgroup 1A and causes rice sheath blight. The predicted protein product of RsPG3 contained 239 amino acid (aa) residues, with a molecular mass of 25.0 kD, whereas RsPG4 encoded a deduced protein of 345 aa residues, with a mass of 37.5 kD. Sequence alignment, phylogenetic analysis and gene ontology indicated that RsPG3 and RsPG4 had endo-PG and exo-PG activity, respectively. Recombinant RsPG3 and RsPG4 both exhibited PG activity that led to the destruction of rice sheaths and release of reducing sugars. Pathogenicity assays showed that the two PGs induced tissue necrosis in rice sheaths, indicating that both RsPG3 and RsPG4 are important virulence factors in the R. solani–rice interaction. Further studies are underway to more clearly define the role of these enzymes in rice cell wall degradation and their interaction with PG inhibitor proteins.

Selection of Differentially Expressed Genes Using the Transcriptome Analysis of Ripening Grape Berries in Response to High Temperature

Purpose: The transcriptome of ‘Campbell Early’ grape berries exposed to high temperature was analyzed to select useful genes by evaluating their differential expression through sequencing short reads on Solexa platform.Research Method: We assembled 118 million high-quality trimmed reads using Velvet followed by Oases.Findings: Functional categorization of expressed transcripts revealed the conservation of genes involved in various biological processes like responses to chemical (12.7%), responses to abiotic stimulus (11.8%), biosynthesis processes (11.8%), and cellular metabolic processes (10.4%) in grape berries exposed to high temperature. The major up-regulated genes included isocitratelyase, cysteine proteinases superfamily protein, cupin family protein, and glycosyl hydrolase genes, and the major down-regulated genes included flavanone 3-hydroxylase, phenylalanine ammonia lyase, chlorophyll A-B binding family protein, and polygalacturonase inhibiting protein genes in grape berries exposed to high temperature. Among genes related to grape coloration, expressions of chalcone and stilbene synthase, flavanone 3-hydroxylase, leucoanthocyanidin dioxygenase, phenylalanine ammonia lyasegenes were more strongly inhibited in berries kept at 35°C than 25°C.Originality/Value: Transcript sets of grape berries generated in this study will provide a resource for gene discovery and development of functional molecular markers to be used in elucidating the mechanism of tolerance to high temperature.

Damage-associated responses of the host contribute to defence against cyst nematodes but not root-knot nematodes.

When nematodes invade and subsequently migrate within plant roots, they generate cell wall fragments (in the form of oligogalacturonides; OGs) that can act as damage-associated molecular patterns and activate host defence responses. However, the molecular mechanisms mediating damage responses in plant-nematode interactions remain unexplored. Here, we characterized the role of a group of cell wall receptor proteins in Arabidopsis, designated as polygalacturonase-inhibiting proteins (PGIPs), during infection with the cyst nematode Heterodera schachtii and the root-knot nematode Meloidogyne incognita. PGIPs are encoded by a family of two genes in Arabidopsis, and are involved in the formation of active OG elicitors. Our results show that PGIP gene expression is strongly induced in response to cyst nematode invasion of roots. Analyses of loss-of-function mutants and overexpression lines revealed that PGIP1 expression attenuates infection of host roots by cyst nematodes, but not root-knot nematodes. The PGIP1-mediated attenuation of cyst nematode infection involves the activation of plant camalexin and indole-glucosinolate pathways. These combined results provide new insights into the molecular mechanisms underlying plant damage perception and response pathways during infection by cyst and root-knot nematodes, and establishes the function of PGIP in plant resistance to cyst nematodes.

Novel Alleles of Two Tightly Linked Genes Encoding Polygalacturonase-Inhibiting Proteins (VrPGIP1 and VrPGIP2) Associated with the Br Locus That Confer Bruchid (Callosobruchus spp.) Resistance to Mungbean (Vigna radiata) Accession V2709

Nearly all mungbean cultivars are completely susceptible to seed bruchids (Callosobruchus chinensis and Callosobruchus maculatus). Breeding bruchid-resistant mungbean is a major goal in mungbean breeding programs. Recently, we demonstrated in mungbean (Vigna radiata) accession V2802 that VrPGIP2, which encodes a polygalacturonase inhibiting protein (PGIP), is the Br locus responsible for resistance to C. chinensis and C. maculatus. In this study, mapping in mungbean accession V2709 using a BC11F2 population of 355 individuals revealed that a single major quantitative trait locus, which controlled resistance to both C. chinensis and C. maculatus, was located in a 237.35 Kb region of mungbean chromosome 5 that contained eight annotated genes, including VrPGIP1 (LOC106760236) and VrPGIP2 (LOC106760237). VrPGIP1 and VrPGIP2 are located next to each other and are only 27.56 Kb apart. Sequencing VrPGIP1 and VrPGIP2 in “V2709” revealed new alleles for both VrPGIP1 and VrPGIP2, named VrPGIP1-1 and VrPGIP2-2, respectively. VrPGIP2-2 has one single nucleotide polymorphism (SNP) at position 554 of wild type VrPGIP2. This SNP is a guanine to cystine substitution and causes a proline to arginine change at residue 185 in the VrPGIP2 of “V2709”. VrPGIP1-1 has 43 SNPs compared with wild type and “V2802”, and 20 cause amino acid changes in VrPGIP1. One change is threonine to proline at residue 185 in VrPGIP1, which is the same as in VrPGIP2. Sequence alignments of VrPGIP2 and VrPGIP1 from “V2709” with common bean (Phaseolus vulgaris) PGIP2 revealed that residue 185 in VrPGIP2 and VrPGIP1 contributes to the secondary structures of proteins that affect interactions between PGIP and polygalacturonase, and that some amino acid changes in VrPGIP1 also affect interactions between PGIP and polygalacturonase. Thus, tightly linked VrPGIP1 and VrPGIP2 are the likely genes at the Br locus that confer bruchid resistance in mungbean “V2709”.

Independent Preharvest Applications of Methyl Jasmonate and Chitosan Elicit Differential Upregulation of Defense-Related Genes with Reduced Incidence of Gray Mold Decay during Postharvest Storage of Fragaria chiloensis Fruit.

The Chilean strawberry (Fragaria chiloensis) fruit has interesting organoleptic properties, but its postharvest life is affected by gray mold decay caused by Botrytis cinerea. The effect of preharvest applications of methyl jasmonate (MeJA) or chitosan on the molecular defense-related responses and protection against gray mold decay were investigated in Chilean strawberry fruit during postharvest storage. Specifically, we inoculated harvested fruit with B. cinerea spores and studied the expression of genes encoding for the pathogenesis-related (PR) proteins β-1,3-glucanases (FcBG2-1, FcBG2-2 and FcBG2-3) and chitinases (FcCHI2-2 and FcCHI3-1), and for polygalacturonase inhibiting proteins (FcPGIP1 and FcPGIP2) at 0, 2, 24, 48, and 72 h post inoculation (hpi). Remarkably, MeJA- and chitosan-treated fruit exhibited a lower incidence of B. cinerea infection than the control-treated at 48 and 72 hpi. At the molecular level, both are efficient elicitors for priming in F. chiloensis fruit since we observed an upregulation of the FcBG2-1, FcBG2-3, FcPGIP1, and FcPGIP2 at 0 hpi. Moreover, a chitosan-mediated upregulation of FcPGIPs at early times post inoculation (2–24 hpi) and MeJA upregulated FcBGs (24–72 hpi) and FcPGIP1 at later times could contribute to reduce B. cinerea incidence by differential upregulation of defense genes. We concluded that preharvest applications of MeJA or chitosan had a long-lasting effect on the reduction of B. cinerea incidence during postharvest as well as an enhancer effect on the induction of PR and PGIP gene expression.

Genetic resources of chili pepper (Capsicum annuum L.) against Phytophthora capsici and their induction through various biotic and abiotic factors

Phytophthora capsici is the most destructive pathogen of vegetables that has blown a serious threat to chili pepper plants. We discussed the control of Phytophthora capsici through manipulation of genetic architecture of chili plant. Various genes encoding transcriptional regulatory and defense related putative proteins such as pathogen-related protein (PR), Anti-Microbial peptides (AMPs), Polygalacturonase-inhibiting proteins (PGIPs), Lipid Transfer Protein (LTP), Pectin Methylesterase (PME), Leucine-rich repeat proteins (LRRs), Osmotin-like and Thaumatin-like protein, have been identified in Capsicum. We also discussed the enhanced resistance to P. capsici infection by treatment with a variety of abiotic and biotic inducers that act on defense signaling pathways involved in disease resistance. We highlighted the vulnerability of chili crop with reference to its genetic resources against Phytophthora blight. Disease control through chemicals is becoming problematic, so we proposed other ways to control the disease severity.

Antifungal genes expressed in transgenic pea (Pisum sativum L.) do not affect root colonization of arbuscular mycorrhizae fungi.

Genetically modified crops have raised concerns about unintended consequences on non-target organisms including beneficial soil associates. Pea transformed with four antifungal genes 1-3 β glucanase, endochitinase, polygalacturonase-inhibiting proteins, and stilbene synthase is currently under field-testing for efficacy against fungal diseases in Canada. Transgenes had lower expression in the roots than leaves in greenhouse experiment. To determine the impact of disease-tolerant pea or gene products on colonization by non-target arbuscular mycorrhizae and nodulation by rhizobium, a field trial was established. Transgene insertion, as single gene or stacked genes, did not alter root colonization by arbuscular mycorrhiza fungus (AMF) or root nodulation by rhizobium inoculation in the field. We found no effect of transgenes on the plant growth and performance although, having a dual inoculant with both AMF and rhizobium yielded higher fresh weight shoot-to-root ratio in all the lines tested. This initial risk assessment of transgenic peas expressing antifungal genes showed no deleterious effect on non-target organisms.

Identification of Fusarium Moniliformin PG Inhibitor using Comparitive Bioinformatics Studies of PGIP of Pharsalus vulgaris and its Neighbours of LRR Superfamily

Invasion of the Phytopathogenic organisms such as fungi, bacteria, virus and nematodes which disrupt the cell wall of the plant by secreting their pectin degrading PolyGalacturonase (PG) enzyme is evaded by the interaction of plant PolyGalacturonase-Inhibiting Proteins (PGIPs). PGIP from Pharsalus vulgaris is reported to blocks the attack of many pathogens, most importantly Fusarium moniliforme. The two PGIP isoforms, PGIP1 and PGIP2 sharing 99% sequence identity show differential activity towards FmPG. PGIP2 blocks FmPG while PGIP1 is unable to do so, while both these proteins belong to the same multigame family. PG-inhibitory activity is also shown by PGIPs of other plant species. Identification of sequence homolog’s of PvPGIP2, sequence similarity of at least 50%, through BLAST followed by MSA and nearest neighbour analysis suggests Glycine max(Soybean) and Pisum sativum(Pea) to be close relatives of Pharsalus vulgaris (Kidney Bean). As already known GmPGIP3 effectively evades infection caused by Fusarium moniliforme blocking its PG enzyme while in Pea the efficacy of PGIP1 against FmPG is yet to be explored. Pairwise sequence comparisons and structure modeling of PvPGIP1 and PsPGIP1 is followed by prediction of putative binding sites of the fungal enzyme FmPG by CASTp. A comparative analysis is then drawn by docking the three PGIP proteins from Kidney Bean (PvPGIP2 and PvPGIP1) and Pea (PsPGIP1) respectively with FmPG using GRAMM-X server. It brings into light that PsPGIP1-FmPG shows same mode of binding as PvPGIP2-FmPG complex despite of being less similar at sequence level. Further on, the residues involved in hydrophobic and ionic interaction are calculated for all the three docked complexes involving FmPG and PGIP proteins from Pharsalus vulgaris (PvPGIP1 and PvPGIP2) and Pisum sativum(PsPGIP1) by PIC. The docking results reveal that PsPGIP1 from Pisum sativum blocks the active cleft of the fungal enzyme FmPG inhibiting the enzyme in similar way as shown by PGIP2 from Pharsalusvulgarisalso the outcomes obtained from the interaction analysis suggest that since same residues of the enzyme FmPG are involved in hydrophobic and ionic interactions in the FmPG-PvPGIP2 complex and FmPG-PsPGIP1 thus PsPGIP1 from Pisum sativumis able to block Polygalacturonase enzyme (FmPG) from Fusarium moniliformin.

Molecular evidence for the involvement of a polygalacturonase-inhibiting protein, GhPGIP1, in enhanced resistance to Verticillium and Fusarium wilts in cotton

Polygalacturonase-inhibiting protein (PGIP), belonging to a group of plant defence proteins, specifically inhibits endopolygalacturonases secreted by pathogens. Herein, we showed that purified GhPGIP1 is a functional inhibitor of Verticillium dahliae and Fusarium oxysporum f. sp. vasinfectum, the two fungal pathogens causing cotton wilt. Transcription of GhPGIP1 was increased in cotton upon infection, wounding, and treatment with defence hormone and H2O2. Resistance by GhPGIP1 was examined by its virus-induced gene silencing in cotton and overexpression in Arabidopsis. GhPGIP1-silenced cotton was highly susceptible to the infections. GhPGIP1 overexpression in transgenic Arabidopsis conferred resistance to the infection, accompanied by enhanced expression of pathogenesis-related proteins (PRs), isochorismate synthase 1 (ICS1), enhanced disease susceptibility 1 (EDS1), and phytoalexin-deficient 4 (PAD4) genes. Transmission electron microscopy revealed cell wall alteration and cell disintegration in plants inoculated with polygalacturonase (PGs), implying its role in damaging the cell wall. Docking studies showed that GhPGIP1 interacted strongly with C-terminal of V. dahliae PG1 (VdPG1) beyond the active site but weakly interacted with C-terminal of F. oxysporum f. sp. vasinfectum (FovPG1). These findings will contribute towards the understanding of the roles of PGIPs and in screening potential combat proteins with novel recognition specificities against evolving pathogenic factors for countering pathogen invasion.

PvPGIP2 Accumulation in Specific Floral Tissues But Not in the Endosperm Limits Fusarium graminearum Infection in Wheat

Fusarium head blight (FHB) caused by Fusarium graminearum is one of the most destructive fungal diseases of wheat worldwide. The pathogen infects the spike at flowering time and causes severe yield losses, deterioration of grain quality, and accumulation of mycotoxins. The understanding of the precise means of pathogen entry and colonization of floral tissue is crucial to providing effective protection against FHB. Polygalacturonase (PG) inhibiting proteins (PGIPs) are cell-wall proteins that inhibit the activity of PGs, a class of pectin-depolymerizing enzymes secreted by microbial pathogens, including Fusarium spp. The constitutive expression of a bean PGIP (PvPGIP2) limits FHB symptoms and reduces mycotoxin accumulation in wheat grain. To better understand which spike tissues play major roles in limiting F. graminearum infection, we explored the use of PvPGIP2 to defend specific spike tissues. We show here that the simultaneous expression of PvPGIP2 in lemma, palea, rachis, and anthers reduced FHB symptoms caused by F. graminearum compared with symptoms in infected nontransgenic plants. However, the expression of PvPGIP2 only in the endosperm did not affect FHB symptom development, indicating that once the pathogen has reached the endosperm, inhibition of the pathogen's PG activity is not effective in preventing its further spread.

Pyramiding PvPGIP2 and TAXI-III But Not PvPGIP2 and PMEI Enhances Resistance Against Fusarium graminearum.

Plant protein inhibitors counteract the activity of cell wall-degrading enzymes (CWDEs) secreted by pathogens to breach the plant cell-wall barrier. Transgenic plants expressing a single protein inhibitor restrict pathogen infections. However, since pathogens secrete a number of CWDEs at the onset of infection, we combined more inhibitors in a single wheat genotype to reinforce further the cell-wall barrier. We combined polygalacturonase (PG) inhibiting protein (PGIP) and pectin methyl esterase inhibitor (PMEI), both controlling the activity of PG, one of the first CWDEs secreted during infection. We also pyramided PGIP and TAXI-III, a xylanase inhibitor that controls the activity of xylanases, key factors for the degradation of xylan, a main component of cereal cell wall. We demonstrated that the pyramiding of PGIP and PMEI did not contribute to any further improvement of disease resistance. However, the presence of both pectinase inhibitors ensured a broader spectrum of disease resistance. Conversely, the PGIP and TAXI-III combination contributed to further improvement of Fusarium head blight (FHB) resistance, probably because these inhibitors target the activity of different types of CWDEs, i.e., PGs and xylanases. Worth mentioning, the reduction of FHB symptoms is accompanied by a reduction of deoxynivalenol accumulation with a foreseen great benefit to human and animal health.

Genomic characterization of plant cell wall degrading enzymes and in silico analysis of xylanases and polygalacturonases of Fusarium virguliforme.

BackgroundPlant cell wall degrading enzymes (PCWDEs) are a subset of carbohydrate-active enzymes (CAZy) produced by plant pathogens to degrade plant cell walls. To counteract PCWDEs, plants release PCWDEs inhibitor proteins (PIPs) to reduce their impact. Several transgenic plants expressing exogenous PIPs that interact with fungal glycoside hydrolase (GH)11-type xylanases or GH28-type polygalacturonase (PG) have been shown to enhance disease resistance. However, many plant pathogenic Fusarium species were reported to escape PIPs inhibition. Fusarium virguliforme is a soilborne pathogen that causes soybean sudden death syndrome (SDS). Although the genome of F. virguliforme was sequenced, there were limited studies focused on the PCWDEs of F. virguliforme. Our goal was to understand the genomic CAZy structure of F. viguliforme, and determine if exogenous PIPs could be theoretically used in soybean to enhance resistance against F. virguliforme.ResultsF. virguliforme produces diverse CAZy to degrade cellulose and pectin, similar to other necrotorphic and hemibiotrophic plant pathogenic fungi. However, some common CAZy of plant pathogenic fungi that catalyze hemicellulose, such as GH29, GH30, GH44, GH54, GH62, and GH67, were deficient in F. virguliforme. While the absence of these CAZy families might be complemented by other hemicellulases, F. virguliforme contained unique families including GH131, polysaccharide lyase (PL) 9, PL20, and PL22 that were not reported in other plant pathogenic fungi or oomycetes. Sequence analysis revealed two GH11 xylanases of F. virguliforme, FvXyn11A and FvXyn11B, have conserved residues that allow xylanase inhibitor protein I (XIP-I) binding. Structural modeling suggested that FvXyn11A and FvXyn11B could be blocked by XIP-I that serves as good candidate for developing transgenic soybeans. In contrast, one GH28 PG, FvPG2, contains an amino acid substitution that is potentially incompatible with the bean polygalacturonase-inhibitor protein II (PvPGIP2).ConclusionsIdentification and annotation of CAZy provided advanced understanding of genomic composition of PCWDEs in F. virguliforme. Sequence and structural analyses of FvXyn11A and FvXyn11B suggested both xylanases were conserved in residues that allow XIP-I inhibition, and expression of both xylanases were detected during soybean roots infection. We postulate that a transgenic soybean expressing wheat XIP-I may be useful for developing root rot resistance to F. virguliforme.Electronic supplementary materialThe online version of this article (doi:10.1186/s12866-016-0761-0) contains supplementary material, which is available to authorized users.

Wound induced Beta vulgaris polygalacturonase-inhibiting protein genes encode a longer leucine-rich repeat domain and inhibit fungal polygalacturonases

Polygalacturonase-inhibiting proteins (PGIPs) are leucine-rich repeat (LRR) proteins involved in plant defense. Three sugar beet (Beta vulgaris L.) PGIP genes, BvPGIP1, BvPGIP2 and BvPGIP3, were isolated from breeding line F1016 and BvPGIP2 and BvPGIP3 from F1010. Full-length cDNA sequences of the three BvPGIP genes encoded a 382 or 384 amino acid peptide. They shared closest similarity with five PGIP-like sequences found in the sugar beet reference genome (RefBeet 1.2.1) as well as Bv(FC607)PGIP1 and Bv(FC607)PGIP2 from sugar beet breeding line FC607. The predicted structure of BvPGIP1, 2 and 3 peptides was that of Phaseolus vulgaris PvPGIP2 with the exception that the sugar beet peptides were about 40 amino acids longer and encoded an additional LRR repeat. RT-PCR and qPCR analyses demonstrated BvPGIP gene expression in leaves, petioles, hypocotyls and highest expression in roots. Leaf and root BvPGIP transcripts increased in response to insect feeding and mechanical wounding. An agarose radial diffusion assay showed that BvPGIP protein extracts inhibited polygalacturonases (PGs) of Rhizoctonia solani, Fusarium oxysporum f. sp. gladioli and F. oxysporum f. sp. betae with the exception that PGIP extracts from line F1010 did not inhibit R. solani PGs. These findings identify sugar beet PGIP genes that encode LRR domains that are longer than reported for most other species. In addition, the wound up-regulated BvPGIP gene expression and inhibition of fungal PGs by PGIP extracts suggest that like other plant PGIPs, the sugar beet genes may play an active role in defense responses.

A gene encoding a polygalacturonase-inhibiting protein (PGIP) is a candidate gene for bruchid (Coleoptera: bruchidae) resistance in mungbean (Vigna radiata).

The Br locus confers bruchid resistance in mungbean; VrPGIP2 (encoding a polygalacturonase inhibitor) is a strong candidate gene for this resistance. The VrPGIP2 sequence differs between resistant and susceptible lines. Azuki bean weevil (Callosobruchus chinensis) and cowpea weevil (Callosobruchus maculatus) are serious insect pests of mungbean during storage. Bruchid resistance in mungbean is controlled by a single dominant locus, Br. Although the Br locus has been located on a genetic map, molecular basis and function of the gene remain unknown. In this study, high-resolution mapping using a BC11F2 population of 418 plants derived from a cross between 'Kamphaeng Saen 1' (KPS1; susceptible) and 'V2802' (resistant) using simple sequence repeat (SSR) markers delimited the Br locus to a genomic region of 38Kb of chromosome 5 containing two annotated genes. EST-SSR marker DMB-SSR158 co-segregated perfectly with the Br locus. Bioinformatics analyses revealed that DMB-SSR158 corresponds to a gene encoding a polygalacturonase inhibitor (polygalacturonase-inhibiting protein PGIP) and was designated as VrPGIP2. Comparison of VrPGIP2 coding sequences between four bruchid-resistant (V2802, V1128, V2817 and TC1966) and four bruchid-susceptible (KPS1, Sulu-1, CM and an unknown accession) mungbean lines revealed six single nucleotide polymorphisms (SNPs) between the resistant and susceptible groups. Three of the six SNPs resulted in amino acid changes; namely, alanine (A) to serine (S) at position 320, leucine (L) to proline (P) at position 332, and threonine (T) to P at position 335 of the VrPGIP2 sequence in resistant lines, compared with that in susceptible lines. The A to S change at position 320 may affect the interaction between PGIP and polygalacuronase. These results indicate that VrPGIP2 is very likely the gene at the Br locus responsible for bruchid resistance in mungbean.

Quantitative Proteomics Reveals the Flooding-Tolerance Mechanism in Mutant and Abscisic Acid-Treated Soybean.

Flooding negatively affects the growth of soybean, and several flooding-specific stress responses have been identified; however, the mechanisms underlying flooding tolerance in soybean remain unclear. To explore the initial flooding tolerance mechanisms in soybean, flooding-tolerant mutant and abscisic acid (ABA)-treated plants were analyzed. In the mutant and ABA-treated soybeans, 146 proteins were commonly changed at the initial flooding stress. Among the identified proteins, protein synthesis-related proteins, including nascent polypeptide-associated complex and chaperonin 20, and RNA regulation-related proteins were increased in abundance both at protein and mRNA expression. However, these proteins identified at the initial flooding stress were not significantly changed during survival stages under continuous flooding. Cluster analysis indicated that glycolysis- and cell wall-related proteins, such as enolase and polygalacturonase inhibiting protein, were increased in abundance during survival stages. Furthermore, lignification of root tissue was improved even under flooding stress. Taken together, these results suggest that protein synthesis- and RNA regulation-related proteins play a key role in triggering tolerance to the initial flooding stress in soybean. Furthermore, the integrity of cell wall and balance of glycolysis might be important factors for promoting tolerance of soybean root to flooding stress during survival stages.

Cloning, expression analysis and recombinant expression of a gene encoding a polygalacturonase-inhibiting protein from tobacco, Nicotiana tabacum

Polygalacturonase inhibiting proteins (PGIPs) are major defensive proteins produced by plant cell walls that play a crucial role in pathogen resistance by reducing polygalacturonase (PG) activity. In the present study, a novel PGIP gene was isolated from tobacco (Nicotiana tabacum), hereafter referred as NtPGIP. A full-length NtPGIP cDNA of 1,412 bp with a 186 bp 5′-untranslated region (UTR), and 209 bp 3′-UTR was cloned from tobacco, NtPGIP is predicted to encode a protein of 338 amino acids. The NtPGIP sequence from genomic DNA showed no introns and sequence alignments of NtPGIP’s deduced amino acid sequence showed high homology with known PGIPs from other plant species. Moreover, the putative NtPGIP protein was closely clustered with several Solanaceae PGIPs. Further, the expression profile of NtPGIP was examined in tobacco leaves following stimulation with the oomycete Phytophthora nicotianae and other stressors, including salicylic acid (SA), abscisic acid (ABA), salt, and cold treatment. The results showed that all of the treatments up-regulated the expression of NtPGIP at different times. To understand the biochemical activity of NtPGIP gene, a full-length NtPGIP cDNA sequence was subcloned into a pET28a vector and transformed into E. coli BL21 (DE3). Recombinant proteins were successfully induced by 1.0 nmol/L IPTG and the purified proteins effectively inhibited Phytophthora capsici PG activity. The results of this study suggest that NtPGIP may be a new candidate gene with properties that could be exploited in plant breeding.