Identification of a chitinase-modifying protein from Fusarium verticillioides: truncation of a host resistance protein by a fungalysin metalloprotease

Chitinase-modifying proteins (cmps) are proteases secreted by fungal pathogens that truncate the plant class IV chitinases ChitA and ChitB during maize ear rot. cmp activity has been characterized for Bipolaris zeicola and Stenocarpella maydis, but the identities of the proteases are not known. Here, we report that cmps are secreted by multiple species from the genus Fusarium, that cmp from Fusarium verticillioides (Fv-cmp) is a fungalysin metalloprotease, and that it cleaves within a sequence that is conserved in class IV chitinases. Protein extracts from Fusarium cultures were found to truncate ChitA and ChitB in vitro. Based on this activity, Fv-cmp was purified from F. verticillioides. N-terminal sequencing of truncated ChitA and MALDI-TOF-MS analysis of reaction products showed that Fv-cmp is an endoprotease that cleaves a peptide bond on the C-terminal side of the lectin domain. The N-terminal sequence of purified Fv-cmp was determined and compared with a set of predicted proteins, resulting in its identification as a zinc metalloprotease of the fungalysin family. Recombinant Fv-cmp also truncated ChitA, confirming its identity, but had reduced activity, suggesting that the recombinant protease did not mature efficiently from its propeptide-containing precursor. This is the first report of a fungalysin that targets a nonstructural host protein and the first to implicate this class of virulence-related proteases in plant disease.     

Crystallographic structure of ChitA,a glycoside hydrolase family 19, plant class IV chitinase from Zea mays

Maize ChitA chitinase is composed of a small, hevein‐like domain attached to a carboxy‐terminal chitinase domain. During fungal ear rot, the hevein‐like domain is cleaved by secreted fungal proteases to produce truncated forms of ChitA. Here, we report a structural and biochemical characterization of truncated ChitA (ChitA ΔN), which lacks the hevein‐like domain. ChitA ΔN and a mutant form (ChitA ΔN‐EQ) were expressed and purified; enzyme assays showed that ChitA ΔN activity was comparable to the full‐length enzyme. Mutation of Glu62 to Gln (ChitA ΔN‐EQ) abolished chitinase activity without disrupting substrate binding, demonstrating that Glu62 is directly involved in catalysis. A crystal structure of ChitA ΔN‐EQ provided strong support for key roles for Glu62, Arg177, and Glu165 in hydrolysis, and for Ser103 and Tyr106 in substrate binding. These findings demonstrate that the hevein‐like domain is not needed for enzyme activity. Moreover, comparison of the crystal structure of this plant class IV chitinase with structures from larger class I and II enzymes suggest that class IV chitinases have evolved to accommodate shorter substrates.     

Truncation of class IV chitinases from Arabidopsis by secreted fungal proteases

Plant class IV chitinases have a small amino‐terminal chitin‐binding domain and a larger chitinase domain, and are involved in plant defence against fungal infection. Our previous work on the chitinases ChitA and ChitB from the model monocotyledon Zea mays showed that the chitin‐binding domain is removed by secreted fungal proteases called fungalysins. In this article, we extend this work to dicotyledons. The effects of fungalysin‐like proteases on four class IV chitinases from the model dicotyledon Arabidopsis thaliana were analysed. Four Arabidopsis chitinases were heterologously expressed in Pichia pastoris, purified and shown to have chitinase activity against a chitohexaose (dp6) substrate. The incubation of these four chitinases with Fv‐cmp, a fungalysin protease secreted by Fusarium verticillioides, resulted in the truncation of AtchitIV3 and AtchitIV5. Moreover, incubation with secreted proteins from Alternaria brassicae, a pathogen of A. thaliana and brassica crops, also led to a similar truncation of AtchitIV3 and AtchitIV4. Our finding that class IV chitinases from both dicotyledons (A. thaliana) and monocotyledons (Z. mays) are truncated by proteases secreted by specialized pathogens of each plant suggests that this may be a general mechanism of plant–fungal pathogenicity.     

Recognition of corn defense chitinases by fungal polyglycine hydrolases

Polyglycine hydrolases (PGH)s are secreted fungal endoproteases that cleave peptide bonds in the polyglycine interdomain linker of ChitA chitinase, an antifungal protein from domesticated corn (Zea mays ssp. mays). These target‐specific endoproteases are unusual because they do not cut a specific peptide bond but select one of many Gly‐Gly bonds within the polyglycine region. Some Gly‐Gly bonds are cleaved frequently while others are never cleaved. Moreover, we have previously shown that PGHs from different fungal pathogens prefer to cleave different Gly‐Gly peptide bonds. It is not understood how PGHs selectively cleave the ChitA linker, especially because its polyglycine structure lacks peptide sidechains. To gain insights into this process we synthesized several peptide analogs of ChitA to evaluate them as potential substrates and inhibitors of Es‐cmp, a PGH from the plant pathogenic fungus Epicoccum sorghi. Our results showed that part of the PGH recognition site for substrate chitinases is adjacent to the polyglycine linker on the carboxy side. More specifically, four amino acid residues were implicated, each spaced four residues apart on an alpha helix. Moreover, analogous peptides with selective Gly‐>sarcosine (N‐methylglycine) mutations or a specific Ser‐>Thr mutation retained inhibitor activity but were no longer cleaved by PGH. Additonally, our findings suggest that peptide analogs of ChitA that inhibit PGH activity could be used to strengthen plant defenses.     

Isolation and characterization of genes encoding two chitinase enzymes from Serratia marcescens

Analysis of clones isolated from a cosmid DNA library indicates that the Serratia marcescens chromosome contains at least two genes, chiA and chiB, which encode distinct secreted forms of the enzyme chitinase. These genes have been characterized by inspection of chitinase activity and secreted proteins in Escherichia coli strains containing subclones of these cosmids. The two chitinase genes show no detectable homology to each other. DNA sequence analysis of one of the genes predicts an amino acid sequence with an N-terminal signal peptide typical of genes encoding secreted bacterial proteins. This gene was mutagenized by cloning a neomycin phosphotransferase gene within its coding region, and the insertion mutation was recombined into the parental S. marcescens strain. The resulting chiA mutant transconjugant showed reduced chitinase production, reduced inhibition of fungal spore germination and reduced biological control of a fungal plant pathogen.     

Fungus- and wound-induced accumulation of mRNA containing a class II chitinase of the pathogenesis-related protein 4 (PR-4) family of maize

Pathogenesis-related (PR) proteins are plant proteins that are induced in response to pathogen attack. PR proteins are grouped into independent families based on their sequences and properties. The PR-4 family comprises class I and class II chitinases. We have isolated a full-length cDNA encoding a chitinase from maize which shares a high degree of nucleotide and amino acid sequence homology with the class II chitinases of the PR-4 family of PR proteins. Our results indicate that fungal infection, and treatment either with fungal elicitors or with moniliformin, a mycotoxin produced by the fungus Fusarium moniliforme, increase the level of ZmPR4 mRNA. In situ mRNA hybridization analysis in sections obtained from fungus-infected germinating embryos revealed that ZmPR4 mRNA accumulation occurs in those cell types that first establish contact with the pathogen. ZmPR4 mRNA accumulation is also stimulated by treatment with silver nitrate whereas the application of the hormones gibberellic acid or acetylsalicylic acid has no effect. Wounding, or treatment with abscisic acid or methyl jasmonate, results in accumulation of ZmPR4 mRNA in maize leaves. Furthermore, the ZmPR4 protein was expressed in Escherichia coli, purified and used to obtain polyclonal antibodies that specifically recognized ZmPR4 in protein extracts from fungus-infected embryos. Accumulation of ZmPR4 mRNA in fungus-infected maize tissues was accompanied by a significant accumulation of the corresponding protein. The possible implications of these findings as part of the general defence response of maize plants against pathogens are discussed.     

Chitinase from Paracoccidioides brasiliensis: molecular cloning, structural, phylogenetic, expression and activity analysis

A full-length cDNA encoding a chitinase (Pbcts1) was cloned by screening a cDNA library from the yeast cells of Paracoccidioides brasiliensis. The cDNA consists of 1888 bp and encodes an ORF of 1218 bp corresponding to a protein of 45 kDa with 406 amino acid residues. The deduced PbCTS1 is composed of two signature family 18 catalytic domains and seems to belong to fungal/bacterial class. Phylogenetic analysis of PbCTS1 and other chitinases suggests the existence of paralogs of several chitinases to be grouped based on specialized functions, which may reflect the multiple and diverse roles played by fungi chitinases. Glycosyl hydrolase activity assays demonstrated that P. brasiliensis is able to produce and secrete these enzymes mainly during transition from yeast to mycelium. The fungus should be able to use chitin as a carbon source. The presence of an endocytic signal in the deduced protein suggests that it could be secreted by a vesicular nonclassical export pathway. The Pbcts1 expression in mycelium, yeast, during differentiation from mycelium to yeast and in yeast cells obtained from infected mice suggests the relevance of this molecule in P. brasiliensis electing PbCTS1 as an attractive drug target.     

Induction of maize acid phosphatase activities under phosphorus starvation

Large variation in phosphorus-(P) acquisition efficiency exists among maize inbred and hybrid genotypes. Acid phosphatases are a type of enzyme that affects P acquisition and P-use efficiency in plants. The objectives of this research were (1) to characterize acid phosphatase activity in maize grown hydroponically under P starvation, and (2) to determine if there is differential induction of acid phosphatases in two maize genotypes previously characterized as P efficient (Mo17) and P inefficient (B73). B73 and Mo17 seedlings were grown hydroponically and both intracellular and secreted acid phosphatase activities were characterized. Fresh seedling weight of both genotypes decreased under P starvation, but percent fresh weight allocated to roots increased 14 days after P starvation in B73. Soluble protein concentration in shoots and roots was affected little, but secreted protein decreased by 40 and 20% in B73 and Mo17 seedlings grown without P for 14 days. Intracellular and secreted acid phosphate activity increased substantially in leaves and roots in B73 and Mo17 in response to P starvation. Secreted APase activity per unit protein increased 310 and 300% in B73 and Mo17, respectively, 7 days after P withdrawal. One of the minor isozymes identified on non-denaturing PAGE, was increased specifically in response to P starvation in both maize genotypes. The patterns and levels of change in APase activities in B73 and Mo17 were not sufficiently different to account for the diverse growth response of these genotypes in low-P conditions. The results suggest that APases may not be a major mechanism for scavenging or acquiring P and changes in APases may reflect a state of P stress in both varieties. Other factors such as root architecture, secretion of low-molecular weight carboxylates and microbial interactions might explain the difference between these two genotypes.     

Expression of a novel chitinase by the fungal endophyte in Poa ampla

Many wild and cultivated cool-season grass species are naturally infected with fungal endophytes of the genera Neotyphodium and Epichlo?. These associations generally are considered mutualistic with the plants benefiting from reduced herbivory and the fungi benefiting from nutrients supplied by the plants. The fungi secrete proteins that might have a role in the interspecies symbiosis. In the interaction between Poa ampla Merr. and the endophyte Neotyphodium sp., a fungal chitinase was detected in the apoplastic protein fraction. The chitinase was also the major protein secreted in culture. Sequence analysis of the chitinase revealed it has a low level of amino acid sequence identity to other fungal chitinases and one of the conserved active site residues is altered. DNA gel-blot analysis indicated the chitinase was encoded by a single gene. Expression of similar chitinases also was detected in endophyte-infected tall fescue (Festuca arundinacea Schreb.), perennial ryegrass (Lolium perenne L.) and Chewings fescue (Festuca rubra L. subsp. fallax [Thuill] Nyman). This is the first report of an endophyte chitinase expressed in the infected host grass. As a secreted hydrolytic enzyme, the chitinase might have roles in the nutrition, growth or defense of the endophyte.     

Transgenic tomato plants expressing a wheat endochitinase gene demonstrate enhanced resistance to <Emphasis Type="Italic">Fusarium oxysporum</Emphasis> f. sp. <Emphasis Type="Italic">lycopersici</Emphasis>

Various chitinases have been shown to inhibit the growth of fungal pathogens in in vitro as well as in planta conditions. chi194, a wheat chitinases gene encoding a 33-kDa chitinase protein, was overexpressed in tomato plants (cv. Pusa Ruby) under the control of maize ubiquitin 1 promoter. The integration of transgene in tomato plants was confirmed with polymerase chain reaction (PCR) and Southern blot analysis. The inheritance of the transgene in T₁ and T₂ generations were shown by molecular analysis and the hygromycin sensitivity test. The broad range of chitinase activity was observed among the transgenic lines in T₀ and a similar range was retained in the T₁ and T₂ generations. Most importantly, the transgenic tomato lines with high chitinase activity were found to be highly resistant to the fungal pathogen Fusarium oxysporum f. sp. lycopersici. Thus, the results demonstrated that the expression of the wheat endochitinase chi194 in tomato plants confers resistance against Fusarium wilt disease caused by the fungal pathogen Fusarium oxysporum f. sp. lycopersici.     

Reduced proteolysis of secreted gelatin and Yps1-mediated alpha-factor leader processing in a Pichia pastoris kex2 disruptant

Heterologous proteins secreted by yeast and fungal expression hosts are occasionally degraded at basic amino acids. We cloned Pichia pastoris homologs of the Saccharomyces cerevisiae basic residue-specific endoproteases Kex2 and Yps1 to evaluate their involvement in the degradation of a secreted mammalian gelatin. Disruption of the P. pastoris KEX2 gene prevented proteolysis of the foreign protein at specific monoarginylic sites. The S. cerevisiae alpha-factor preproleader used to direct high-level gelatin secretion was correctly processed at its dibasic site in the absence of the prototypical proprotein convertase Kex2. Disruption of the YPS1 gene had no effect on gelatin degradation or processing of the alpha-factor propeptide. When both the KEX2 and YPS1 genes were disrupted, correct precursor maturation no longer occurred. The different substrate specificities of both proteases and their mutual redundancy for propeptide processing indicate that P. pastoris kex2 and yps1 single-gene disruptants can be used for the alpha-factor leader-directed secretion of heterologous proteins otherwise degraded at basic residues.     

Preparative two-dimensional polyacrylamide gel electrophoresis of rat liver ribosomal proteins and determination of their amino acid compositions

A maize root fraction which inactivates nitrate reductase has been shown to have protease activity which can be measured by the hydrolysis of azocasein. This inactivating enzyme was also found to inactivate yeast tryptophan synthase. Yeast proteases A and B, which inactivate this latter enzyme, also gave a specific inactivation of the maize nitrate reductase. The maize root inactivating enzyme, like yeast protease B, degraded casein, and was inhibited by phenylmethylsulphonyl fluoride. A partially-purified yeast inhibitor prevented catalysis by the yeast proteases and maize root inactivating enzyme, but purified yeast inhibitors were without effect on the latter protein. The level of nitrate reductase-inactivating activity, and associated azocasein-degrading activity, increased with age of the maize root. Evidence was obtained for a heat stable inhibitor which maintained them in an inactive state, especially in the young root tip cells.     

A Phytophthora infestans cystatin-like protein targets a novel tomato papain-like apoplastic protease

There is emerging evidence that the proteolytic machinery of plants plays important roles in defense against pathogens. The oomycete pathogen Phytophthora infestans, the agent of the devastating late blight disease of tomato (Lycopersicon esculentum) and potato (Solanum tuberosum), has evolved an arsenal of protease inhibitors to overcome the action of host proteases. Previously, we described a family of 14 Kazal-like extracellular serine protease inhibitors from P. infestans. Among these, EPI1 and EPI10 bind and inhibit the pathogenesis-related (PR) P69B subtilisin-like serine protease of tomato. Here, we describe EPIC1 to EPIC4, a new family of P. infestans secreted proteins with similarity to cystatin-like protease inhibitor domains. Among these, the epiC1 and epiC2 genes lacked orthologs in Phytophthora sojae and Phytophthora ramorum, were relatively fast-evolving within P. infestans, and were up-regulated during infection of tomato, suggesting a role during P. infestans-host interactions. Biochemical functional analyses revealed that EPIC2B interacts with and inhibits a novel papain-like extracellular cysteine protease, termed Phytophthora Inhibited Protease 1 (PIP1). Characterization of PIP1 revealed that it is a PR protein closely related to Rcr3, a tomato apoplastic cysteine protease that functions in fungal resistance. Altogether, this and earlier studies suggest that interplay between host proteases of diverse catalytic families and pathogen inhibitors is a general defense-counterdefense process in plant-pathogen interactions.     

Removal of Vacuolar Targeting Signal from Class I Vacuolar Chitinase leads to its Extracellular Secretion in Transgenic Tobacco

Chitinase, an antifungal pathogenesis related (PR) protein is present in different isoforms. Class I basic chitinase which is generally more antifungal in nature compared to other chitinase classes, is present in vacuoles. It is speculated that extracellular secretion of this vacuolar enzyme by removing its vacuolar targeting signal at C- terminus might further increase its effectivity. Tobacco class I chitinase cDNA was earlier modified by PCR to add two stop codons before vacuolar targeting signal, so that the protein without this signal would be secreted extracellularly.Transgenic tobacco plants were raised with modified chitinase cDNA and native unmodified cDNA, both under the control of CaMV 35 S promoter, using Agrobacterium mediated transformation. Transgenic plants with unmodified class I chitinase cDNA expressed the enzyme in vacuoles and those having modified cDNA expressed the enzyme in extracellular spaces while retaining its biological activity.