Plant Polygalacturonase-inhibiting Proteins Research Articles

New Players in the Interaction Between Beetle Polygalacturonases and Plant Polygalacturonase-Inhibiting Proteins: Insights From Proteomics and Gene Expression Analyses.

Plants possess various defense strategies to counter attacks from microorganisms or herbivores. For example, plants reduce the cell-wall-macerating activity of pathogen- or insect-derived polygalacturonases (PGs) by expressing PG-inhibiting proteins (PGIPs). PGs and PGIPs belong to multi-gene families believed to have been shaped by an evolutionary arms race. The mustard leaf beetle Phaedon cochleariae expresses both active PGs and catalytically inactive PG pseudoenzymes. Previous studies demonstrated that (i) PGIPs target beetle PGs and (ii) the role of PG pseudoenzymes remains elusive, despite having been linked to the pectin degradation pathway. For further insight into the interaction between plant PGIPs and beetle PG family members, we combined affinity purification with proteomics and gene expression analyses, and identified novel inhibitors of beetle PGs from Chinese cabbage (Brassica rapa ssp. pekinensis). A beetle PG pseudoenzyme was not targeted by PGIPs, but instead interacted with PGIP-like proteins. Phylogenetic analysis revealed that PGIP-like proteins clustered apart from “classical” PGIPs but together with proteins, which have been involved in developmental processes. Our results indicate that PGIP-like proteins represent not only interesting novel PG inhibitor candidates in addition to “classical” PGIPs, but also fascinating new players in the arms race between herbivorous beetles and plant defenses.

Interaction between Brassica napus polygalacturonase inhibition proteins and Sclerotinia sclerotiorum polygalacturonase: implications for rapeseed resistance to fungal infection.

BnPGIPs interacted with Sclerotinia sclerotiorum PGs to improve rapeseed SSR resistance at different levels; the BnPGIP-overexpression lines did not affect plant morphology or seed quality traits. Plant polygalacturonase-inhibiting proteins (PGIPs) play a crucial role in plant defence against phytopathogenic fungi by inhibiting fungal polygalacturonase (PG) activity. We overexpressed BnPGIP2, BnPGIP5, and BnPGIP10 genes in an inbred line 7492 of rapeseed (Brassica napus). Compared with 7492WT, the overexpression of BnPGIP2 lines significantly increased Sclerotinia sclerotiorum resistance in both seedlings and adult plants. BnPGIP5 overexpression lines exhibited decreased S. sclerotiorum disease symptoms in seedlings only, whereas BnPGIP10 overexpression lines did not improve Sclerotinia resistance for seedlings or adult plants. Quantitative real-time PCR analysis of S. sclerotiorum PG1, SsPG3, SsPG5, and SsPG6 genes in overexpressing BnPGIP lines showed that these pathogenic genes in the Sclerotinia resistance transgenic lines exhibited low expression in stem tissues. Split-luciferase complementation experiments confirmed the following: BnPGIP2 interacts with SsPG1 and SsPG6 but not with SsPG3 or SsPG5; BnPGIP5 interacts with SsPG3 and SsPG6 but not with SsPG1 or SsPG5; and BnPGIP10 interacts with SsPG1 but not SsPG3, SsPG5, or SsPG6. Leaf crude protein extracts from BnPGIP2 and BnPGIP5 transgenic lines displayed high inhibitory activity against the SsPG crude protein. BnPGIP-overexpression lines with Sclerotinia resistance displayed a lower accumulation of H2O2 and higher expression of the H2O2-removing gene BnAPX (ascorbate peroxidase) than 7492WT, as well as elevated expression of defence response genes including jasmonic acid/ethylene and salicylic acid pathways after S. sclerotiorum infection. The plants overexpressing BnPGIP exhibited no difference in either agronomic traits or grain yield from 7492WT. This study provides potential target genes for developing S. sclerotiorum resistance in rapeseed.

Direct evidence for a new mode of plant defense against insects via a novel polygalacturonase-inhibiting protein expression strategy

Plant cell wall-associated polygalacturonase-inhibiting proteins (PGIPs) are widely distributed in the plant kingdom. They play a crucial role in plant defense against phytopathogens by inhibiting microbial polygalacturonases (PGs). PGs hydrolyze the cell wall polysaccharide pectin and are among the first enzymes to be secreted during plant infection. Recent studies demonstrated that herbivorous insects express their own PG multi-gene families, raising the question whether PGIPs also inhibit insect PGs and protect plants from herbivores. Preliminary evidence suggested that PGIPs may negatively influence larval growth of the leaf beetle Phaedon cochleariae (Coleoptera: Chrysomelidae) and identified BrPGIP3 from Chinese cabbage (Brassica rapa ssp. pekinensis) as a candidate. PGIPs are predominantly studied in planta because their heterologous expression in microbial systems is problematic and instability and aggregation of recombinant PGIPs has complicated in vitro inhibition assays. To minimize aggregate formation, we heterologously expressed BrPGIP3 fused to a glycosylphosphatidylinositol (GPI) membrane anchor, immobilizing it on the extracellular surface of insect cells. We demonstrated that BrPGIP3_GPI inhibited several P. cochleariae PGs in vitro, providing the first direct evidence of an interaction between a plant PGIP and an animal PG. Thus, plant PGIPs not only confer resistance against phytopathogens, but may also aid in defense against herbivorous beetles.

Amino acid substitutions in a polygalacturonase inhibiting protein (OsPGIP2) increases sheath blight resistance in rice

BackgroundAn economic strategy to control plant disease is to improve plant defense to pathogens by deploying resistance genes. Plant polygalacturonase inhibiting proteins (PGIPs) have a vital role in plant defense against phytopathogenic fungi by inhibiting fungal polygalacturonase (PG) activity. We previously reported that rice PGIP1 (OsPGIP1) inhibits PG activity in Rhizoctonia solani, the causal agent of rice sheath blight (SB), and is involved in regulating resistance to SB.ResultHere, we report that OsPGIP2, the protein ortholog of OsPGIP1, does not possess PGIP activity; however, a few amino acid substitutions in a derivative of OsPGIP2, of which we provide support for L233F being the causative mutation, appear to impart OsPGIP2 with PG inhibition capability. Furthermore, the overexpression of mutated OsPGIP2L233F in rice significantly increased the resistance of transgenic lines and decreased SB disease rating scores. OsPGIP2L233F transgenic lines displayed an increased ability to reduce the tissue degradation caused by R. solani PGs as compared to control plants. Rice plants overexpressing OsPGIP2L233F showed no difference in agronomic traits and grain yield as compared to controls, thus demonstrating its potential use in rice breeding programs.ConclusionsIn summary, our results provide a new target gene for breeding SB resistance through genome-editing or natural allele mining.

Mutation of key amino acids in the polygalacturonase‐inhibiting proteins CkPGIP1 and GhPGIP1 improves resistance to Verticillium wilt in cotton

Verticillium wilt, one of the most devastating diseases of cotton (Gossypium hirsutum), causes severe yield and quality losses. Given the effectiveness of plant polygalacturonase-inhibiting proteins (PGIPs) in reducing fungal polygalacturonase (PG) activity, it is necessary to uncover the key functional amino acids to enhance cotton resistance to Verticillium dahliae. To identify novel antifungal proteins, the selectivity of key amino acids was investigated by screening against a panel of relevant PG-binding residues. Based on the obtained results, homologous models of the mutants were established. The docking models showed that hydrogen bonds and structural changes in the convex face in the conserved portion of leucine-rich repeats (LRRs) may be essential for enhanced recognition of PG. Additionally, we successfully constructed Cynanchum komarovii PGIP1 (CkPGIP1) mutants Asp176Val, Pro249Gln, and Asp176Val/Pro249Gln and G.hirsutum PGIP1 (GhPGIP1) mutants Glu169Val, Phe242Gln, and Glu169Val/Phe242Gln with site-directed mutagenesis. The proteins of interest can effectively inhibit VdPG1 activity and V.dahliae mycelial growth in a dose-dependent manner. Importantly, mutants that overproduced PGIP in Arabidopsis and cotton showed enhanced resistance to V.dahliae, with reduced Verticillium-associated chlorosis and wilting. Furthermore, the lignin content was measured in mutant-overexpressing plants, and the results showed enhanced lignification of the xylem, which blocked the spread of V.dahliae. Thus, using site-directed mutagenesis assays, we showed that mutations in CkPGIP1 and GhPGIP1 give rise to PGIP versatility, which allows evolving recognition specificities for PG and is required to promote Verticillium resistance in cotton by restricting the growth of invasive fungal pathogens.

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.

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.

Polygalacturonase‐inhibiting proteins – leucine‐rich repeat proteins in plant defence

Plant polygalacturonase-inhibiting proteins (PGIPs) belong to the leucine-rich repeat (LRR) family and are known to prevent pathogen invasion by inhibiting the plant cell wall degrading enzyme, polygalacturonase. Our study reveals that these multigene-encoded defence proteins found in flowering plants only exhibit identical domain architecture with 10 tandemly-arranged LRRs. This implies that variations of PGIP inhibitory properties are not associated with the number of the repeats but with subtle changes in the sequence content of the repeats. The first and eighth repeat contain more mutations compared to the strict conservation of the plant-specific LRRs or any repeat at other positions. Each of these repeats forms a separate cluster in the phylogenetic tree, both within and across plant families, thus suggesting uniqueness with respect to their position. A study of the genes encoding PGIPs, shows the existence of two categories (i) single exon and hence no intron; and (ii) two exons with an intron in between. Analyses of the intron phase and correlation of the exon-intron structure with the compact structural modules in PGIPs support insertion of introns in the pre-existing single exon genes and thus the intron late model. Lack of conservation of phase across families and formation of individual clusters for each family in the phylogenetic tree drawn with the intron sequences illustrate the event of insertion that took place separately in each of these families.

Integration of evolutionary and desolvation energy analysis identifies functional sites in a plant immunity protein

Plant immune responses often depend on leucine-rich repeat receptors that recognize microbe-associated molecular patterns or pathogen-specific virulence proteins, either directly or indirectly. When the recognition is direct, a molecular arms race takes place where plant receptors continually and rapidly evolve in response to virulence factor evolution. A useful model system to study ligand-receptor coevolution dynamics at the protein level is represented by the interaction between pathogen-derived polygalacturonases (PGs) and plant polygalacturonase-inhibiting proteins (PGIPs). We have applied codon substitution models to PGIP sequences of different eudicotyledonous families to identify putative positively selected sites and then compared these sites with the propensity of protein surface residues to interact with protein partners, based on desolvation energy calculations. The 2 approaches remarkably correlated in pinpointing several residues in the concave face of the leucine-rich repeat domain. These residues were mutated into alanine and their effect on the recognition of several PGs was tested, leading to the identification of unique hotspots for the PGIP-PG interaction. The combined approach used in this work can be of general utility in cases where structural information about a pattern-recognition receptor or resistance-gene product is available.

A single amino acid substitution in highly similar endo-PGs from Fusarium verticillioides and related Fusarium species affects PGIP inhibition

Endo-polygalacturonase (PG) may be a critical virulence factor secreted by several fungi upon plant invasion. The single-copy gene encoding PG in Fusarium verticillioides and in eight other species of the Gibberella fujikuroi complex (F. sacchari, F. fujikuroi, F. proliferatum, F. subglutinans, F. thapsinum, F. nygamai, F. circinatum, and F. anthophilum) was functionally analyzed in this paper. Both the nucleotide and amino acid sequences were highly similar among the 12 strains of F. verticillioides analyzed, as well as among those from the G. fujikuroi complex. The PGs were not inhibited by the polygalacturonase-inhibiting proteins (PGIPs) from the monocot asparagus and leek plants, but were inhibited to variable extents by bean PGIP. PGs from F. verticillioides, F. nygamai and one strain of F. proliferatum were barely inhibited. Residue 97 within PG was demonstrated to contribute to the different levels of inhibition. Together these findings provide new insights into the structural and functional relationships between the PG from the species of the G. fujikuroi complex and the plant PGIP.

Crystal structure of the endopolygalacturonase from the phytopathogenic fungus Colletotrichum lupini and its interaction with polygalacturonase‐inhibiting proteins

Crystal structure of the endopolygalacturonase from the phytopathogenic fungus <i>Colletotrichum lupini</i> and its interaction with polygalacturonase‐inhibiting proteins

Fungal pathogens can elude the inhibition by host plant polygalacturonase inhibiting proteins (PGIPs) by different mechanisms.

To infect and colonize successfully host plants, fungal pathogens have evolved specific strategies. Many fungi overcome the defensive barrier represented by the cell wall by producing large amount of cell wall degrading enzymes (CWDE) during infection. Among CWDE, pectinases, and especially endo-polygalacturonases (endo- PG), play an important role since they depolymerise the pectic component of the cell wall and middle lamella. Polygalacturonase-inhibiting proteins (PGIPs) are plant defence molecules able to block the polygalacturonase activity of fungal pathogens. Therefore, PGIP could be used both in traditional and innovative breeding programmes to defeat those pathogens that express endo-PG activity to assist with plant infection. However, fungi have evolved strategies to overcome PGIP inhibition. For example, Sclerotinia sclerotiorum in the early stage of soybean infection produces considerable amount of a basic PG encoded by the Sspgb gene. By quantitative RT-PCR it was shown that, initially, the transcript of this pg gene largely overcomes that of the soybean pgip gene. Therefore, at this stage, the PGIP level would be not enough to significantly counteract fungal PG activity.

An endo-polygalacturonase (PG) of Fusarium moniliforme escaping inhibition by plant polygalacturonase-inhibiting proteins (PGIPs) provides new insights into the PG–PGIP interaction

Polygalacturonase-inhibiting proteins (PGIPs) are plant defence molecules inhibiting the activity of fungal endo-polygalacturonases (endo-PGs). We found that soybean and bean PGIPs inhibited the endo-PG activity produced by the isolate FC-10 of Fusarium moniliforme but not the enzyme activity produced by the isolate PD of F. moniliforme. The bean PGIP proved to be ineffective against all the PG isoforms produced by the PD isolate. Deduced amino acid sequence comparison between PGs from PD, FC-10 and 62264 isolates identified the structural regions of the enzyme possibly related to its resistance to PGIP inhibition. These include one region at the N-terminal portion of the enzyme and a few single amino acid substitutions along the entire sequence, two of which surrounding the active site.

CDNA cloning and expression of polygalacturonaseinhibiting proteins (PGIPs) from red raspberry (Rubus idaeus)

Plant polygalacturonase-inhibiting proteins (PGIPs) inhibit endo-polygalacturonases (endo-PGs) released by invasive fungi. Two potential PGlP cDNAs have been cloned from raspberry (Rubus idaeus). PGIP1 corresponds to a full-length PGIP cDNA with a high degree of identity with previously isolated genes and maintains all the characteristic features of PGIP peptides. A genomic fragment of PGIP1 identifies a single 243 bp intron which is efficiently spliced out of the PGIP1 premRNA transcript. PGIP2 contains a frame shift mutation which would lead to the loss of 105 amino acids off the C-terminus. Contrary to PGIP activity levels in raspberry (Johnston et al., 1993), expression analysis detected PGIP1 at equal levels throughout fruit development from closed flower, through to ripe fruit.