Polygalacturonase-inhibiting proteins in defense against phytopathogenic fungi

Polygalacturonase-inhibiting proteins (PGIPs) are ubiquitous plant cell wall proteins that are directed against fungal polygalacturonases (PGs), which are important pathogenicity factors. The inhibiting activity of PGIPs directly reduces the aggressive potential of PGs. In addition, it causes PGs to form more long-chain oligogalacturonides that are able to induce defense responses, thereby indirectly contributing to the plant defense. Recent evidence demonstrates that PGIPs are efficient defense proteins and limit fungal invasion. PGIPs and the products of many plant resistance genes share a leucine-rich repeat (LRR) structure, which provides specific recognition of pathogen-derived molecules. The high level of polymorphism of both PGIPs and polygalacturonases is an invaluable tool for deciphering the structure, function and evolution of plant LRR proteins and their ligands. Furthermore, information about PGIP structure and evolution paves the way to the development of efficient strategies for crop protection.     

果实表达PGIPs的基因克隆及功能研究进展

多聚半乳糖醛酸酶(PGs)是病原真菌早期侵染植物的一个重要致病因子。多聚半乳糖醛酸酶抑制蛋白(PGIPs)作为植物防御蛋白,能特异性抑制真菌分泌的多聚半乳糖醛酸酶,并通过延长寡聚半乳糖醛酸(OGs)的稳定期激活植物防御反应。综述PGIPs在植物细胞中的定位,PGIPs与PGs之间的作用方式,PGIPs基因的分离与克隆,以及PGIPs对果实感病的影响,并对PGIPs的研究前景进行展望。     

多聚半乳糖醛酸酶抑制蛋白的结构、功能以及应用研究进展

真菌病害严重影响植物的生长发育。为了自我保护,植物进化出了许多抵御病原真菌入侵的策略,例如防御相关蛋白的产生。多聚半乳糖醛酸酶抑制蛋白（polygalacturonase-inhibiting proteins,PGIPs）是近年来研究较多的一种植物防御蛋白,它能与真菌分泌的多聚半乳糖醛酸酶（polygalacturonases,PGs）特异性结合,降低PGs水解植物细胞壁的活性并在植物体内累积能激活多种防御反应的长链寡聚半乳糖醛酸（oligogalacturonides,OGs）,从而达到抑制真菌侵染的目的。主要介绍了PGIPs的结构、功能及其抗菌机理,并综述了PGIPs在国内外转基因抗病育种中的应用研究进展。     

Tandemly duplicated Arabidopsis genes that encode polygalacturonase-inhibiting proteins are regulated coordinately by different signal transduction pathways in response to fungal infection

Polygalacturonase-inhibiting proteins (PGIPs) are plant proteins that counteract fungal polygalacturonases, which are important virulence factors. Like many other plant defense proteins, PGIPs are encoded by gene families, but the roles of individual genes in these families are poorly understood. Here, we show that in Arabidopsis, two tandemly duplicated PGIP genes are upregulated coordinately in response to Botrytis cinerea infection, but through separate signal transduction pathways. AtPGIP2 expression is mediated by jasmonate and requires COI1 and JAR1, whereas AtPGIP1 expression is upregulated strongly by oligogalacturonides but is unaffected by salicylic acid, jasmonate, or ethylene. Both AtPGIP1 and AtPGIP2 encode functional inhibitors of polygalacturonase from Botrytis, and their overexpression in Arabidopsis significantly reduces Botrytis disease symptoms. Therefore, gene duplication followed by the divergence of promoter regions may result in different modes of regulation of similar defensive proteins, thereby enhancing the likelihood of defense gene activation during pathogen infection.     

Plants use identical inhibitors to protect their cell wall pectin against microbes and insects

As fundamentally different as phytopathogenic microbes and herbivorous insects are, they enjoy plant‐based diets. Hence, they encounter similar challenges to acquire nutrients. Both microbes and beetles possess polygalacturonases (PGs) that hydrolyze the plant cell wall polysaccharide pectin. Countering these threats, plant proteins inhibit PGs of microbes, thereby lowering their infection rate. Whether PG‐inhibiting proteins (PGIPs) play a role in defense against herbivorous beetles is unknown. To investigate the significance of PGIPs in insect–plant interactions, feeding assays with the leaf beetle Phaedon cochleariae on Arabidopsis thaliana pgip mutants were performed. Fitness was increased when larvae were fed on mutant plants compared to wild‐type plants. Moreover, PG activity was higher, although PG genes were downregulated in larvae fed on PGIP‐deficient plants, strongly suggesting that PGIPs impair PG activity. As low PG activity resulted in delayed larval growth, our data provide the first in vivo correlative evidence that PGIPs act as defense against insects.     

Polygalacturonase inhibiting proteins: players in plant innate immunity?

Polygalacturonase-inhibiting proteins (PGIPs) are extracellular leucine-rich repeat (LRR) proteins that recognize and inhibit fungal polygalacturonases (PGs). The PG-PGIP interaction favours the accumulation of elicitor-active oligogalacturonides and causes the activation of defence responses. Small gene families encode PGIP isoforms that differ in affinity and specificity for PGs secreted by different pathogens. The consensus motif within the LRR structure of PGIPs is the same as that of the extracellular receptors of the plant innate immune system. Structural and functional evidence suggest that PGIPs are versatile proteins involved in innate immunity and that they are capable of recognizing different surface motifs of functionally related but structurally variable PGs.     

Role of extracytoplasmic leucine rich repeat proteins in plant defence mechanisms

Plant-pathogen interactions involve highly complex series of reactions in disease development. Plants are endowed with both, resistance and defence genes. The activation of defence genes after contact with avirulence gene products of pathogens depends on signals transduced by leucine-rich repeats (LRRs) contained in resistance genes. Additionally, LRRs play roles for various actions following ligand recognition. Polygalacturonase inhibiting proteins (PGIPs), the only plant LRR protein with known ligands, are pectinase inhibitors, bound by ionic interactions to the extracellular matrix (ECM) of plant cells. They have a high affinity for fungal endopolygalacturonases (endoPGs). PGIP genes are organised in families encoding proteins with similar physical characteristics but different specificities. They are induced by infection and stress related signals. The molecular basis of PG-PGIP interaction serves as a model to understand the evolution of plant LRR proteins in recognising non-self-molecules. Extensins form a different class of structural proteins with repetitive sequences. They are also regulated by wounding and pathogen infection. Linkage of extensins with LRR motifs is highly significant in defending host tissues against pathogen invasion. Overexpression of PGIPs or expression of several PGIPs in a plant tissue, and perhaps manipulation of extensin expression could be possible strategies for disease management.     

Fungal polygalacturonases exhibit different substrate degradation patterns and differ in their susceptibilities to polygalacturonase-inhibiting proteins.

Polygalacturonic acid (PGA) was hydrolyzed by polygalacturonases (PGs) purified from six fungi. The oligogalacturonide products were analyzed by HPAEC-PAD (high performance anion exchange chromatography-pulsed amperimetric detection) to assess their relative amounts and degrees of polymerization. The abilities of the fungal PGs to reduce the viscosity of a solution of PGA were also determined. The potential abilities of four polygalacturonase-inhibiting proteins (PGIPs) from three plant species to inhibit or to modify the hydrolytic activity of the fungal PGs were determined by colorimetric and HPAEC-PAD analyses, respectively. Normalized activities of the different PGs acting upon the same substrate resulted in one of two distinct oligogalacturonide profiles. Viscometric analysis of the effect of PGs on the same substrate also supports two distinct patterns of cleavage. A wide range of susceptibility of the various PGs to inhibition by PGIPs was observed. The four PGs that were inhibited by all PGIPs tested exhibited an endo/exo mode of substrate cleavage, while the three PGs that were resistant to inhibition by one or more of the PGIPs proceed by a classic endo pattern of cleavage.     

Cloning and functional analysis of three genes encoding polygalacturonase-inhibiting proteins from Capsicum annuum and transgenic CaPGIP1 in tobacco in relation to increased resistance to two fungal pathogens

Polygalacturonase-inhibiting proteins (PGIPs) are plant cell wall glycoproteins that can inhibit fungal endopolygalacturonases (PGs). The PGIPs directly reduce the aggressive potential of PGs. Here, we isolated and functionally characterized three members of the pepper (Capsicum annuum) PGIP gene family. Each was up-regulated at a different time following stimulation of the pepper leaves by Phytophthora capcisi and abiotic stresses including salicylic acid, methyl jasmonate, abscisic acid, wounding and cold treatment. Purified recombinant proteins individually inhibited activity of PGs produced by Alternaria alternata and Colletotrichum nicotianae, respectively, and virus-induced gene silencing in pepper conferred enhanced susceptibility to P. capsici. Because three PGIP genes acted similarily in conferring resistance to infection by P. capsici, and because individually purified proteins showed consistent inhibition against PG activity of both pathogens, CaPGIP1 was selected for manipulating transgenic tobacco. The crude proteins from transgenic tobacco exhibited distinct enhanced resistance to PG activity of both fungi. Moreover, the transgenic tobacco showed effective resistance to infection and a significant reduction in the number of infection sites, number of lesions and average size of lesions in the leaves. All results suggest that CaPGIPs may be involved in plant defense response and play an important role in a plant’s resistance to disease.     

Antisense expression of the Arabidopsis thaliana AtPGIP1 gene reduces polygalacturonase-inhibiting protein accumulation and enhances susceptibility to Botrytis cinerea

Polygalacturonases (PGs) hydrolyze the homogalacturonan of plant cell-wall pectin and are important virulence factors of several phytopathogenic fungi. In response to abiotic and biotic stress, plants accumulate PG-inhibiting proteins (PGIPs) that reduce the activity of fungal PGs. In Arabidopsis thaliana, PGIPs with comparable activity against BcPG1, an important pathogenicity factor of the necrotrophic fungus Botrytis cinerea, are encoded by two genes, AtPGIP1 and AtPGIP2. Both genes are induced by fungal infection through different signaling pathways. We show here that transgenic Arabidopsis plants expressing an antisense AtPGIP1 gene have reduced AtPGIP1 inhibitory activity and are more susceptible to B. cinerea infection. These results indicate that PGIP contributes to basal resistance to this pathogen and strongly support the vision that this protein plays a role in Arabidopsis innate immunity.     

Polygalacturonase-inhibiting protein (PGIP) in plant defence: a structural view

Polygalacturonase-inhibiting proteins are plant extracellular leucine-rich repeat proteins that specifically bind and inhibit fungal polygalacturonases. The interaction with PGIP limits the destructive potential of polygalacturonases and might trigger the plant defence responses induced by oligogalacturonides. A high degree of polymorphism is found both in PGs and PGIPs, accounting for the specificity of different plant inhibitors for PGs from different fungi. Here, we review the structural features and our current understanding of the PG-PGIP interaction.     

Directed mutagenesis confirms the functional importance of positively selected sites in polygalacturonase inhibitor protein

Polygalacturonase inhibitor proteins (PGIPs) protect plants against invasion by diverse microbial and invertebrate enemies that use polygalacturonase (PG) to breach the plant cell wall. Directed mutagenesis has identified specific natural mutations conferring novel defensive capability in green bean PGIP against a specific fungal PG. These same sites are identified as positively selected by phylogenetic codon-substitution models, demonstrating the utility of such models for connecting retrospective comparative analyses with contemporary, ecologically relevant variation.     

Transgenic expression of polygalacturonase-inhibiting proteins in Arabidopsis and wheat increases resistance to the flower pathogen Fusarium graminearum

Fusarium head blight (FHB), caused by Fusarium graminearum, is one of the most important diseases of wheat worldwide, resulting in yield losses and mycotoxin contamination. The molecular mechanisms regulating Fusarium penetration and infection are poorly understood. Beside mycotoxin production, cell wall degradation may play a role in the development of FHB. Many fungal pathogens secrete polygalacturonases (PGs) during the early stages of infection, and plants have evolved polygalacturonase-inhibiting proteins (PGIPs) to restrict pectin degradation during fungal infection. To investigate the role of plant PGIPs in restricting the development of FHB symptoms, we first used Arabidopsis thaliana, whose genome encodes two PGIPs (AtPGIP1 and AtPGIP2). Arabidopsis transgenic plants expressing either of these PGIPs under control of the CaMV 35S promoter accumulate inhibitory activity against F.?graminearum PG in their inflorescences, and show increased resistance to FHB. Second, transgenic wheat plants expressing the bean PvPGIP2 in their flowers also had a significant reduction of symptoms when infected with F.?graminearum. Our data suggest that PGs likely play a role in F.?graminearum infection of floral tissues, and that PGIPs incorporated into wheat may be important for increased resistance to FHB.     

A Novel Polygalacturonase-Inhibiting Protein (PGIP) from Lathyrus sativus L. Seeds

Polygalacturonase-inhibiting proteins (PGIPs) are extracellular plant proteins bound to the plant cell wall containing leucine-rich repeats (LRR). They play an important role in plant defence being able to inhibit fungal endopolygalacturonases (EPGs), the first enzymes secreted by phytopathogenic fungi during plant infection. In the present work, a novel PGIP (LsPGIP) has been isolated from Lathyrus sativus seeds. LsPGIP exhibited an inhibitory activity towards EPGs from Aspergillus niger and Rhizopus spp. A pI value of 8.3 and a molecular mass of 40 kDa were determined for the purified inhibitor. Furthermore, N-terminal sequence up to residue 20 revealed that LsPGIP exhibit a high percentage of identity with PGIP from Actinidia deliciosa. A secondary structure similar to those of other polygalacturonase inhibitors was also inferred form circular dichroism data.     

A Single Amino-Acid Substitution Allows Endo-Polygalacturonase of Fusarium verticillioides to Acquire Recognition by PGIP2 from Phaseolus vulgaris

Polygalacturonases (PGs) are secreted by phytopathogenic fungi to degrade the plant cell wall homogalacturonan during plant infection. To counteract Pgs, plants have evolved polygalacturonase-inhibiting proteins (PGIPs) that slow down fungal infection and defend cell wall integrity. PGIPs favour the accumulation of oligogalacturonides, which are homogalacturonan fragments that act as endogenous elicitors of plant defence responses. We have previously shown that PGIP2 from Phaseolus vulgaris (PvPGIP2) forms a complex with PG from Fusarium phyllophilum (FpPG), hindering the enzyme active site cleft from substrate. Here we analyse by small angle X-ray scattering (SAXS) the interaction between PvPGIP2 and a PG from Colletotrichum lupini (CluPG1). We show a different shape of the PG-PGIP complex, which allows substrate entry and provides a structural explanation for the different inhibition kinetics exhibited by PvPGIP2 towards the two isoenzymes. The analysis of SAXS structures allowed us to investigate the basis of the inability of PG from Fusarium verticilloides (FvPG) to be inhibited by PvPGIP2 or by any other known PGIP. FvPG is 92.5% identical to FpPG, and we show here, by both loss- and gain-of-function mutations, that a single amino acid site acts as a switch for FvPG recognition by PvPGIP2.     

Cloning of polygalacturonase inhibitor protein genes from Solanum brevidens Fill

New data were obtained for the Solanum brevidens Fill. nucleotide sequences coding for polygalacturonase inhibitor proteins (PGIPs), which are involved in plant defense against phytopathogenic fungi. Highly degenerate primers directed to the conserved regions of the known PGIP genes of tomato, kiwi, apple, carrot, and grape were used to clone four pgip genes and one pseudogene from the genome of S. brevidens, a species that is closely related to cultivated potato, forms no tubers, is highly resistant to phytopathogens, and is often employed in potato breeding. The sequenced part of the coding region of the new genes is 924 bp and codes for a protein of 308 amino acid residues (without the leader peptide). The genes were designated as pgipSbr1(1), pgipSbr1 (2). pgipSbr2, pgipSbr3, and pgipSbr4. The amino acid sequences of the S. brevidens PGIPs have 90.9-99.4% identity to each other and 94% identity to PGIP of Lycopersicon esculentum Mill., another member of the family Solanaceae. The amino acid residues differing between S. brevidens PGIPs were assumed to determine the selectivity of interactions with particular polyglucuronases of phytopathogenic fungi.     

Polygalacturonase Inhibiting Proteins (PGIPs) of Plants

Polygalacturonase inhibiting proteins (PGIPs) are widely distributed in plants and appear to play an important role in protection of plants from fungal infection and also in maturation and ripening of fruits. They are leucine rich thermostable glycoproteins of about 40 kD and exhibit differential inhibition of polygalacturonases from different sources. A lot of attention is now being paid to these proteins with respect to their physicochemical and biological properties and also to their genes. These proteins show a lot of sequence homology to several signal transduction molecules from diverse species and have been suggested to play a similar role in plants. These proteins are also potentially important from biotechnological perspective. This review focuses on the recent work carried on PGIPs and its implications.