Polygalacturonase-inhibiting Protein Research Articles

Cucumber PGIP2 is involved in resistance to gray mold disease

Polygalacturonase inhibitor protein (PGIP) restricts fungal growth and colonization and functions in plant immunity. Gray mold in cucumber is a common fungal disease caused by Botrytis cinerea, and is widespread and difficult to control in cucumber (Cucumis sativus L.) production. In this study, Cucumis sativus polygalacturonase-inhibiting protein 2 (CsPGIP2) was found to be upregulated in response to gray mold in cucumber. CsPGIP2 was detected in the endoplasmic reticulum, cell membrane, and cell wall after transient transformation of protoplasts and tobacco. A possible interaction between Botrytis cinerea polygalacturonase 3 (BcPG3) and CsPGIP2 was supported by protein interaction prediction and BiFC analysis. Transgenic Arabidopsis plants expressing CsPGIP2 were constructed and exhibited smaller areas of gray mold infection compared to wild type (WT) plants after simultaneous inoculation. Evans blue dye (EBD) confirmed greater damage for WT plants, with more intense dyeing than for the transgenic Arabidopsis. Interestingly, compared to WT, transgenic Arabidopsis exhibited higher superoxide dismutase (AtSOD1) expression, antioxidant enzyme activities, lignin content, net photosynthetic rate (Pn), and photochemical activity. Our results suggest that CsPGIP2 stimulates a variety of plant defense mechanisms to enhance transgenic Arabidopsis resistance against gray mold disease.

Zig, Zag, and ’Zyme: leveraging structural biology to engineer disease resistance

AbstractDynamic host–pathogen interactions determine whether disease will occur. Pathogen effector proteins are central players in such disease development. On one hand, they improve susceptibility by manipulating host targets; on the other hand, they can trigger immunity after recognition by host immune receptors. A major research direction in the study of molecular plant pathology is to understand effector-host interactions, which has informed the development and breeding of crops with enhanced disease resistance. Recent breakthroughs on experiment- and artificial intelligence-based structure analyses significantly accelerate the development of this research area. Importantly, the detailed molecular insight of effector–host interactions enables precise engineering to mitigate disease. Here, we highlight a recent study by Xiao et al., who describe the structure of an effector-receptor complex that consists of a fungal effector, with polygalacturonase (PG) activity, and a plant-derived polygalacturonase-inhibiting protein (PGIP). PGs weaken the plant cell wall and produce immune-suppressive oligogalacturonides (OGs) as a virulence mechanism; however, PGIPs directly bind to PGs and alter their enzymatic activity. When in a complex with PGIPs, PGs produce OG polymers with longer chains that can trigger immunity. Xiao et al. demonstrate that a PGIP creates a new active site tunnel, together with a PG, which favors the production of long-chain OGs. In this way, the PGIP essentially acts as both a PG receptor and enzymatic manipulator, converting virulence to defense activation. Taking a step forward, the authors used the PG-PGIP complex structure as a guide to generate PGIP variants with enhanced long-chain OG production, likely enabling further improved disease resistance. This study discovered a novel mechanism by which a plant receptor plays a dual role to activate immunity. It also demonstrates how fundamental knowledge, obtained through structural analyses, can be employed to guide the design of proteins with desired functions in agriculture.

Polygalacturonase-inhibiting proteins as an exogenously applied natural solution for prevention of postharvest fungal infections

Polygalacturonase inhibiting proteins (PGIPs) are plant proteins involved in the inhibition of polygalacturonases (PGs), cell-wall degrading enzymes often secreted by phytopathogenic fungi. Previously, we confirmed that PGIP2 from Phaseolus vulgaris (PvPGIP2) can inhibit the growth of Aspergillus niger and Botrytis cinerea on agar plate. In this study, we further validated the feasibility of using PGIP as an environmental and ecological friendly agent to prevent fungal infection post-harvest. We found that application of either purified PGIP (full length PvPGIP2 or truncated tPvPGIP2_5–8), or PGIP-secreting Saccharomyces cerevisiae strains can effectively inhibit fungal growth and necrotic lesions on tobacco leaf. We also examined the effective amount and thermostability of PGIP when applied on plants. A concentration of 0.75 mg/mL or higher can significantly reduce the area of B. cinerea lesions. The activity of full-length PvPGIPs is not affected after incubation at various temperatures ranging from −20 to 42 °C for 24 h, while truncated tPvPGIP2_5–8 lost some efficacy after incubation at 42 °C. Furthermore, we have also examined the efficacy of PGIP on tomato fruit. When the purified PvPGIP2 proteins were applied to tomato fruit inoculated with B. cinerea at a concentration of roughly 1.0 mg/mL, disease incidence and area of disease had reduced by more than half compared to the controls without PGIP treatment. This study explores the potential of PGIPs as exogenously applied, eco-friendly fungal control agents on fruit and vegetables post-harvest.

A plant mechanism of hijacking pathogen virulence factors to trigger innate immunity.

Polygalacturonase-inhibiting proteins (PGIPs) interact with pathogen-derived polygalacturonases to inhibit their virulence-associated plant cell wall-degrading activity but stimulate immunity-inducing oligogalacturonide production. Here we show that interaction between Phaseolus vulgaris PGIP2 (PvPGIP2) and Fusarium phyllophilum polygalacturonase (FpPG) enhances substrate binding, resulting in inhibition of the enzyme activity of FpPG. This interaction promotes FpPG-catalyzed production of long-chain immunoactive oligogalacturonides, while diminishing immunosuppressive short oligogalacturonides. PvPGIP2 binding creates a substrate binding site on PvPGIP2-FpPG, forming a new polygalacturonase with boosted substrate binding activity and altered substrate preference. Structure-based engineering converts a putative PGIP that initially lacks FpPG-binding activity into an effective FpPG-interacting protein. These findings unveil a mechanism for plants to transform pathogen virulence activity into a defense trigger and provide proof of principle for engineering PGIPs with broader specificity.

Polygalacturonase inhibiting protein enhances cell wall strength of strawberry fruit for resistance to Botrytis cinerea infection

Polygalacturonase inhibitor protein (PGIP) plays an active role in resisting pathogens from degrading cell walls. Strawberry fruit quality is strongly affected by Botrytis cinerea (B. cinerea), so PGIP genes from heterologous species were cloned and analyzed to identify genes having good B.cinerea infection resistance. In this study, a PGIP gene from grape fruit was isolated and heterologously transiently expressed in strawberry fruit. The results revealed that VvPGIP (PGIPO) overexpression in strawberry fruit led to B. cinerea infection resistance, but RNA interference of VvPGIP (PGIPI) made fruit susceptible to B. cinerea infection. qPCR indicated that expression levels of FaPGIP was increased after transient expression of VvPGIP in PGIPO fruit for 48 h as compared to the control and that polygalacturonase enzyme (PG) was significantly down-regulated, whereas PGIPI fruit had the opposite effect, suggesting that VvPGIP may activate the FaPGIP expression levels, thereby reducing the disease incidence and index of strawberry fruits and improving the disease resistance of the fruits. VvPGIP also inhibited the decrease of total pectin and cellulose content. Transcriptome and metabolome results showed that PGIP is widely involved in photosynthesis, signal transduction, and secondary metabolism of fruits. In addition, many enzyme activities and defense reaction were also significantly enriched. Using Y2H and BiFC technology, we screened and verified the interaction protein of VvPGIP with Endo-1, 3-Beta-Glucanase (VvGLU) and pathogen-related protein 4 (VvPR-4) on the cell membrane. Taken together, PGIP as a key protein in plant defense response functions a key role in regulating the strength of cell walls and can inhibit the degradation of plant cell walls by pathogens.

The polygalacturonase-inhibiting protein PpPGIP1, positively regulates vacuolar invertase activity via a protein-protein interaction with PpVIN2 in peach fruit

The vacuolar invertase (VIN) gene PpVIN2 is induced at chilling temperatures, and plays an important role in sucrose metabolism in peach fruit during cold storage. Using a dual membrane yeast two-hybrid system, we found 22 proteins interacted with PpVIN2. Of these, PpPGIP1 (polygalacturonase-inhibiting protein) is a leucine-rich repeat glycoprotein and sensitives to chilling temperature (5 °C), based on the expression level of which increased significantly accompanied with the chilling injury. Colocalization of PpPGIP1 and PpVIN2 was demonstrated by confocal microscopy and their interaction between was demonstrated by bimolecular fluorescence complementation (BiFC). Transient overexpression of PpPGIP1 in peach fruit increased VIN activity significantly. Transient virus-induced gene silencing of PpPGIP1 decreased VIN activity significantly, which led to decreases sucrose decomposition. PpPGIP1 recombinant protein was successfully expressed by constructing pMAL-c6T-PpPGIP1, and PpPGIP1 increased the VIN activity in vitro. Taken together, PpPGIP1 positively regulates VIN activity via an interaction with PpVIN2; this interaction results in accelerated sucrose decomposition in peaches under chilling temperature. These findings provide a new perspective for understanding the new function of PpPGIP1 related with the rapid decomposition of sucrose and the occurrence of chilling injury in peach fruit during cold storage.

Unraveling the genomic reorganization of polygalacturonase-inhibiting proteins in chickpea.

Polygalacturonase-inhibiting proteins (PGIPs) are cell wall proteins that inhibit pathogen polygalacturonases (PGs). PGIPs, like other defense-related proteins, contain extracellular leucine-rich repeats (eLRRs), which are required for pathogen PG recognition. The importance of these PGIPs in plant defense has been well documented. This study focuses on chickpea (Cicer arietinum) PGIPs (CaPGIPs) owing to the limited information available on this important crop. This study identified two novel CaPGIPs (CaPGIP3 and CaPGIP4) and computationally characterized all four CaPGIPs in the gene family, including the previously reported CaPGIP1 and CaPGIP2. The findings suggest that CaPGIP1, CaPGIP3, and CaPGIP4 proteins possess N-terminal signal peptides, ten LRRs, theoretical molecular mass, and isoelectric points comparable to other legume PGIPs. Phylogenetic analysis and multiple sequence alignment revealed that the CaPGIP1, CaPGIP3, and CaPGIP4 amino acid sequences are similar to the other PGIPs reported in legumes. In addition, several cis-acting elements that are typical of pathogen response, tissue-specific activity, hormone response, and abiotic stress-related are present in the promoters of CaPGIP1, CaPGIP3, and CaPGIP4 genes. Localization experiments showed that CaPGIP1, CaPGIP3, and CaPGIP4 are located in the cell wall or membrane. Transcript levels of CaPGIP1, CaPGIP3, and CaPGIP4 genes analyzed at untreated conditions show varied expression patterns analogous to other defense-related gene families. Interestingly, CaPGIP2 lacked a signal peptide, more than half of the LRRs, and other characteristics of a typical PGIP and subcellular localization indicated it is not located in the cell wall or membrane. The study's findings demonstrate CaPGIP1, CaPGIP3, and CaPGIP4's similarity to other legume PGIPs and suggest they might possess the potential to combat chickpea pathogens.

High Sclerotinia sclerotiorum resistance in rapeseed plant has been achieved by OsPGIP6.

Sclerotinia sclerotiorum, a worldwide distributed fungal pathogen, causes serious adverse effects on the yield and seed quality of rapeseed. Polygalacturonase-inhibiting proteins (PGIPs) can protect the cell wall from degradation by pathogen-secreted polygalacturonases (PGs). The present study found several PGIPs from Oryza sativa, especially OsPGIP6 and 3 have much higher inhibitory activities to SsPGs than BnPGIP2 from Brassica napus. Among them, OsPGIP1, 4, 6 can significantly elevate the resistance of transgenic Arabidopsis to S. sclerotiorum. Subsequently, OsPGIP1, 3, 4, 6 were subjected to SSR resistance assay in transgenic rapeseed plants. Among which, OsPGIP6 showed the highest resistance to S. sclerotiorum. At 48 h after detached leaves inoculation, the lesion area of OE-OsPGIP6 rapeseed plants is only 17.93% of the non-transgenic line, and 22.17, 21.32, 52.78, 56.47%, compared to OE-BnPGIP2, OE-OsPGIP1, OE-OsPGIP2, OE-OsPGIP4, respectively. Furthermore, the lesion area of OE-OsPGIP6 reached 10.11% compared to WT at 72 hpi. Also, the lesion length on the stem of OE-OsPGIP6 plants was reduced by 36.83% compared to WT. These results reveal that OsPGIP family, especially OsPGIP6, has a great potential in rapeseed S. sclerotiorum-resistance breeding.

Tandemly duplicated genes encoding polygalacturonase inhibitors are associated with bruchid (Callosobruchus chinensis) resistance in moth bean (Vigna aconitifolia)

Bruchids are stored-grain insect pests responsible for serious seed loss in legume crops. A previous study using an F2 population (F2OA) derived from a cross between wild moth-bean (Vigna aconitifolia [Jacq.] Maréchal) accession TN67 (resistant) and cultivated moth-bean accession ICPMO056 (susceptible) revealed that resistance to the azuki bean weevil (Callosobruchus chinensis L.) in TN67 was regulated by a single gene located in the major quantitative trait locus—qVacBrc2.1. In this study, qVacBrc2.1 was finely mapped and candidate genes in this locus were identified using F2OA and another large F2 population (F2NB) derived from the cross mentioned previously. In contrast to the previous study, segregation analysis in the F2NB population revealed that resistance against this pest was controlled by two genes. Furthermore, the addition of novel markers to qVacBrc2.1 and reanalysis of the QTL in the F2OA population demonstrated that qVacBrc2.1 constituted two linked QTLs—qVacBrc2.1-A and qVacBrc2.1-B. The presence of qVacBrc2.1-B was verified using the population F2NB. Comparative genomics using three Vigna spp. strongly suggested the presence of two tandemly duplicated genes, VacPGIP1 and VacPGIP2, which encoded polygalacturonase inhibitors (polygalacturonase-inhibiting proteins) as the candidates for conferring resistance, but only VacPGIP1 could be successfully cloned and sequenced. The alignment of VacPGIP1 coding sequences of TN67 and ICPMO056 revealed eight single nucleotide polymorphisms, three of which altered the amino-acid sequence of the predicted domains of polygalacturonase inhibitors in ICPMO056. Overall, these findings indicate that VacPGIP1 and VacPGIP2 regulated C. chinensis resistance in TN67.

Differences in defence-related gene expression and metabolite accumulation reveal insights into the resistance of Greek grape wine cultivars to <i> Botrytis </i> bunch rot

Berries of 13 Greek grape wine cultivars were evaluated for resistance to Botrytis bunch rot. Artificial inoculations on detached berries revealed that the Greek cultivars tested varied regarding their susceptibility to Botrytis cinerea. Cultivar (cv.) “Limnio” was found to be highly resistant, while higher susceptibility was observed on berries of cv. “Roditis”. To determine the molecular basis of the observed resistance or susceptibility of cv. “Limnio” and “Roditis”, an expression analysis of 12 defence-related genes, was carried out on artificially inoculated berries of the two cultivars at different time points after inoculation. Gene expression measurements in the resistant cv. “Limnio” showed that the artificial inoculation with the pathogen triggered the induction of genes encoding pathogenesis-related (PR) proteins such as chitinases (CHIT), polygalacturonase-inhibiting protein (PGIP), serine proteinase inhibitor (PIN) and enzymes involved in phytoalexin synthesis such as phenylalanine ammonia-lyase (PAL) and stilbene synthase (STS). In contrast, on the susceptible cv. “Roditis”, most of the same genes were down-regulated. Metabolomic analysis revealed significant differences in the initial metabolic profiles of “Limnio” and “Roditis” berries. Furthermore, in response to inoculation, the abundance of several metabolites increased in the resistant cultivar indicating intensification of metabolic processes. Proline and mannitol accumulation, as well as the modification of metabolites related to phenylpropanoid and lignin biosynthesis, are among the major players in defence responses of the “Limnio” cultivar. The above findings enhance our understanding of the resistance of Greek grape wine cultivars to B. cinerea and, at the same time, lay the foundation for breeding wine grape cultivars in the future.

A fungal extracellular effector inactivates plant polygalacturonase-inhibiting protein

Plant pathogens degrade cell wall through secreted polygalacturonases (PGs) during infection. Plants counteract the PGs by producing PG-inhibiting proteins (PGIPs) for protection, reversibly binding fungal PGs, and mitigating their hydrolytic activities. To date, how fungal pathogens specifically overcome PGIP inhibition is unknown. Here, we report an effector, Sclerotinia sclerotiorum PGIP-INactivating Effector 1 (SsPINE1), which directly interacts with and functionally inactivates PGIP. S. sclerotiorum is a necrotrophic fungus that causes stem rot diseases on more than 600 plant species with tissue maceration being the most prominent symptom. SsPINE1 enhances S. sclerotiorum necrotrophic virulence by specifically interacting with host PGIPs to negate their polygalacturonase-inhibiting function via enhanced dissociation of PGIPs from PGs. Targeted deletion of SsPINE1 reduces the fungal virulence. Ectopic expression of SsPINE1 in plant reduces its resistance against S. sclerotiorum. Functional and genomic analyses reveal a conserved virulence mechanism of cognate PINE1 proteins in broad host range necrotrophic fungal pathogens.

Proteomics analysis reveals new insights into surface pitting of sweet cherry cultivars displaying contrasting susceptibility

ABSTRACT Surface pitting in sweet cherry (Prunus avium L.) is characterised by depressions development on the skin surface. Pitting damage happens during harvest and postharvest handling and develops during cold storage. This study compared two cultivars with contrasting susceptibility to pitting (Kordia: tolerant; Sweetheart: susceptible) using a LC-MS/MS proteomic approach to identify key metabolic and signalling pathways related to this disorder during ripening and postharvest storage. The variability observed in the principal component analysis was driven by the cultivars suggesting that surface pitting susceptibility is triggered by the inherent differences between cultivars. Proteins involved in anthocyanin biosynthesis were more abundant in Kordia. Moreover, CCR (cinnamoyl-CoA reductase) and FLS (flavonol synthase) proteins showed higher abundance in Sweetheart. An overexpression in enzymes related to the synthesis of abscisic acid (ABA), jasmonic acid (JA) and ethylene were found in Kordia. A higher abundance of sucrose synthase (SUS), UDP-glycosyltransferases (UDP-GT) and polygalacturonase-inhibiting proteins (PGIP) were observed in Kordia, while a higher content of invertase (INV) was observed in Sweetheart. The overexpression of the studied pathways suggests that tolerance to surface pitting could be correlated to one or more of these factors, and susceptibility might be given by the inherent differences in the metabolic processes of each cultivar.

Apple vacuolar processing enzyme 4 is regulated by cysteine protease inhibitor and modulates fruit disease resistance.

Ring rot is a destructive apple disease caused by Botryosphaeria dothidea. The resistance mechanism of apple plants to B. dothidea remains unclear. Here, we show that APPLE VACUOLAR PROCESSING ENZYME 4 (MdVPE4) is involved in resistance to B. dothidea. MdVPE4 silencing reduced fruit disease resistance, whereas its overexpression improved resistance. Gene expression analysis revealed that MdVPE4 influenced the expression of fruit disease resistance-related genes, such as APPLE POLYGALACTURONASE 1 (MdPG1), APPLE POLYGALACTURONASE INHIBITOR PROTEIN 1 (MdPGIP1), APPLE ENDOCHITINASE 1 (MdCHI1), and APPLE THAUMATIN-LIKE PROTEIN 1 (MdTHA1). The expression of the four genes responding to B. dothidea infection decreased in MdVPE4-silenced fruits. Further analysis demonstrated that B. dothidea infection induced MdVPE4 expression and enzyme activation in apple fruits. Moreover, MdVPE4 activity was modulated by apple cysteine proteinase inhibitor 1 (MdCPI1), which also contributed to resistance towards B. dothidea, as revealed by gene overexpression and silencing analysis. MdCPI1 interacted with MdVPE4 and inhibited its activity. However, MdCPI1 expression was decreased by B. dothidea infection. Taken together, our findings indicate that the interaction between MdVPE4 and MdCPI1 plays an important role in modulating fruit disease resistance to B. dothidea.

Expression GWAS of PGIP1 Identifies STOP1-Dependent and STOP1-Independent Regulation of PGIP1 in Aluminum Stress Signaling in Arabidopsis.

To elucidate the unknown regulatory mechanisms involved in aluminum (Al)-induced expression of POLYGALACTURONASE-INHIBITING PROTEIN 1 (PGIP1), which is one of the downstream genes of SENSITIVE TO PROTON RHIZOTOXICITY 1 (STOP1) regulating Al-tolerance genes, we conducted a genome-wide association analysis of gene expression levels (eGWAS) of PGIP1 in the shoots under Al stress using 83 Arabidopsis thaliana accessions. The eGWAS, conducted through a mixed linear model, revealed 17 suggestive SNPs across the genome having the association with the expression level variation in PGIP1. The GWAS-detected SNPs were directly located inside transcription factors and other genes involved in stress signaling, which were expressed in response to Al. These candidate genes carried different expression level and amino acid polymorphisms. Among them, three genes encoding NAC domain-containing protein 27 (NAC027), TRX superfamily protein, and R-R-type MYB protein were associated with the suppression of PGIP1 expression in their mutants, and accordingly, the system affected Al tolerance. We also found the involvement of Al-induced endogenous nitric oxide (NO) signaling, which induces NAC027 and R-R-type MYB genes to regulate PGIP1 expression. In this study, we provide genetic evidence that STOP1-independent NO signaling pathway and STOP1-dependent regulation in phosphoinositide (PI) signaling pathway are involved in the regulation of PGIP1 expression under Al stress.

Recent Advances in Understanding the Function of the PGIP Gene and the Research of Its Proteins for the Disease Resistance of Plants

Polygalacturonase-inhibiting protein (PGIP) is an important plant biochemical anti-disease factor. PGIP has a leucine-rich repeat structure that can selectively bind and inhibit the activity of endo-polygalacturonase (endo-PG) in fungi, playing a key role in plant disease resistance. The regulation of PGIP in plant disease resistance has been well studied, and the effect of PGIP to increase disease resistance is clear. This review summarizes recent advances in understanding the PGIP protein structure, the PGIP mechanism of plant disease resistance, and anti-disease activity by PGIP gene transfer. This overview should contribute to a better understanding of PGIP function and can help guide resistance breeding of PGIP for anti-disease effects.

Endophytic Microorganisms Associated with Reversion of Silverleaf Disease Symptoms in Apple

Silverleaf is caused by the fungus Chondrostereum purpureum, which produces wood necrosis and foliar silvering in woody plants. Field observations and studies in apple have shown the reversion of foliar symptoms. Since plants were clones and received identical agronomical management, it was hypothesized that reversion is driven by endophytic microbiota. Thus, the objectives of this study were to compare healthy, diseased and reverted plants with respect to their physiology, endophytic microbial communities, antagonistic ability of their endophytes against C. purpureum and defense-genes-expression. Water-potential, stomatal-conductance, chlorophyll-content and fluorescence were measured. Endophytic bacterial and fungal DNA were analyzed by DGGE, and communities richness and similarity were calculated. Wood cores were collected and bacterial and fungal endophytes were isolated and confronted with C. purpureum virulent strains in dual-culture assays. Defense-genes-expression were measured by qPCR. Results indicated that there were no differences in physiological parameters between healthy and reverted plants, except for fluorescence, and both type of plants differed from diseased ones. Bacterial and fungal communities richness were similar in healthy and reverted plants, and higher than in diseased ones. Endophytes from reverted and healthy plants showed high antagonism to C. purpureum. Furthermore, NPR1-gene-expression was upregulated in reverted plants, while PAL and PGIP showed higher values in diseased plants. Overall, physiological, molecular and microbial characteristics were similar between healthy and reverted plants, and both differed from diseased ones. Therefore, reversion of symptoms is associated with changes in the endophytic microbiota, which seems to be a promising source of biological control agents against C. purpureum.

Isolation, purification, and characterization of pectin methylesterase inhibitor and polygalacturonase inhibitor protein from Indian lemon (Citrus limon L.)

Proteins acting as powerful inhibitors of plant pectin methylesterase and polygalacturonase were isolated from whole lemon fruits (Citrus limon L.). Pectin methylesterase inhibitor (PMEI) and polygalacturonase inhibitor protein (PGIP) were purified using DEAE Sepharose column, resulting in fold purity of 89.13 and 81.16 and having a molecular mass of 35 and 38 kDa, respectively as estimated using SDS-PAGE and MALDI-TOF mass spectroscopy. The optimum pH of purified PMEI and PGIP was pH 6 and pH 4.5 while the inhibitors showed good stability in the pH range of 5–8 and 3.5 to 5.5, respectively. Both the inhibitors from C. limon demonstrated an optimum temperature of 55 °C. Thermal inactivation data suggested that purified PGIP was more heat stable than PMEI. The inhibition kinetics of PMEI and PGIP towards C. limon PME and C. limon PG was of a non-competitive type. Both PMEI and PGIP obeyed first-order inactivation kinetics. The PMEI and PGIP exhibited different extent of inhibition towards PME and PG from other fruit sources analyzed in this study. As these inhibitors inhibit PME and PG from other plant sources they can be used in fruit-based products to control undesirable endogenous enzyme activities as an alternative to thermal processing.

Exploring the potential of engineering polygalacturonase-inhibiting protein as an ecological, friendly, and nontoxic pest control agent.

In plants, polygalacturonase-inhibiting proteins (PGIPs) play critical roles for resistance to fungal disease by inhibiting the pectin-depolymerizing activity of endopolygalacturonases (PGs), one type of enzyme secreted by pathogens that compromises plant cell walls and leaves the plant susceptible to disease. Here, the interactions between PGIPs from Phaseolus vulgaris (PvPGIP1 and PvPGIP2) and PGs from Aspergillus niger (AnPG2), Botrytis cinerea (BcPG1 and BcPG2), and Fusarium moniliforme (FmPG3) were reconstituted through a yeast two hybrid (Y2H) system to investigate the inhibition efficiency of various PvPGIP1 and 2 truncations and mutants. We found that tPvPGIP2_5-8, which contains LRR5 to LRR8 and is only one-third the size of the full length peptide, exhibits the same level of interactions with AnPG and BcPGs as the full length PvPGIP2 via Y2H. The inhibitory activities of tPvPGIP2_5-8 on the growth of A. niger and B. cinerea were then examined and confirmed on pectin agar. On pectin assays, application of both full length PvPGIP2 and tPvPGIP2_5-8 clearly slows down the growth of A. niger and B. cinerea. Investigation on the sequence-function relationships of PGIP utilizing a combination of site directed mutagenesis and a variety of peptide truncations suggests that LRR5 could have the most essential structural feature for the inhibitory activities, and may be a possible target for the future engineering of PGIP with enhanced activity. This study highlights the potential of plant-derived PGIPs as a candidate for future in planta evaluation as a pest control agent.

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.

Chitin nanofibers trigger membrane bound defense signaling and induce elicitor activity in plants

The present study demonstrated that chitin-based nanofibers (CNFs) trigger the chitinase genes (PGIP1 and CaChi2), while elevating salicylic acid that can protect plants against pathogens. Cross-talk between this genetic induction and salicylic-acid-mediated immune response was also observed, which may arm a plant against multiple pathovars. Crab and mushroom based CNFs were synthesized by electrospinning and ball milling techniques. Plants (mung bean, Vigna radiata) (pepper, Capsicum annuum) were pre-inoculated with CNFs and treated with the pathogens Scrolotium rolfsii for pepper and Macrophomina phaseolina for mung bean and shrimp-based CNFs were used as a control. Treated plants had elevated levels of chitinase genes in response to CNFs at inoculation concentrations <10 mg/mL both in soil and media, to protect them against the pathogenic fungal disease. After 24 h of exposure to the pathogens, qRT-PCR showed genes class II chitinase gene (CaChi2) and polygalacturonase inhibitor protein 1 (PGIP1) to be up-regulated in both root and shoot at 0.1 and 1 mg/mL of inoculation, respectively. The ball milled mushroom CNFs were sufficient to trigger the membrane based enzymes with less diameter (≥15 nm) to be most efficient versus others. In vitro analysis showed IC50 of ball milled mushroom CNFs to be most efficient in limiting the growth of fungal biomass. Further trigger-like effects were prominent in reducing pathogenic fungal spread in both species.