Todd A. Naumann

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

Background: Fusarium fungi manipulate plant defenses to cause disease. Results: Fusarium fungi secrete fungalysin proteases that truncate maize class IV chitinases. Conclusions: Fusarium fungalysins target nonstructural defense proteins associated with plant disease resistance. Significance: Manipulating the fungalysin-chitinase interaction might improve fungal disease resistance of plants. 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.

Tn5 Transposase with an Altered Specificity for Transposon Ends

Tn5 is a composite bacterial transposon that encodes a protein, transposase (Tnp), required for movement of the transposon. The initial step in the transposition pathway involves specific binding of Tnp to 19-bp end recognition sequences. Tn5 contains two different specific end sequences, termed outside end (OE) and inside end (IE). In Escherichia coli, IE is methylated by Dam methylase (IE(ME)). This methylation greatly inhibits recognition by Tnp and greatly reduces the ability of transposase to facilitate movement of IE defined transposons. Through use of a combinatorial random mutagenesis technique (DNA shuffling), we have isolated an IE(ME)-specific hyperactive form of Tnp, Tnp sC7v.2.0, that is able to promote high levels of transposition of IE(ME) defined transposons in vivo and in vitro while functioning at wild-type levels with OE transposons. This protein contains a critical glutamate-to-valine mutation at amino acid 58 that is responsible for this change in end specificity.

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.

Polyglycine hydrolases: Fungal β-lactamase-like endoproteases that cleave polyglycine regions within plant class IV chitinases

Polyglycine hydrolases are secreted fungal proteases that cleave glycine–glycine peptide bonds in the inter‐domain linker region of specific plant defense chitinases. Previously, we reported the catalytic activity of polyglycine hydrolases from the phytopathogens Epicoccum sorghi (Es‐cmp) and Cochliobolus carbonum (Bz‐cmp). Here we report the identity of their encoding genes and the primary amino acid sequences of the proteins responsible for these activities. Peptides from a tryptic digest of Es‐cmp were analyzed by LC‐MS/MS and the spectra obtained were matched to a draft genome sequence of E. sorghi. From this analysis, a 642 amino acid protein containing a predicted β‐lactamase catalytic region of 280 amino acids was identified. Heterologous strains of the yeast Pichia pastoris were created to express this protein and its homolog from C. carbonum from their cDNAs. Both strains produced recombinant proteins with polyglycine hydrolase activity as shown by SDS‐PAGE and MALDI‐MS based assays. Site directed mutagenesis was used to mutate the predicted catalytic serine of Es‐cmp to glycine, resulting in loss of catalytic activity. BLAST searching of publicly available fungal genomes identified full‐length homologous proteins in 11 other fungi of the class Dothideomycetes, and in three fungi of the related class Sordariomycetes while significant BLAST hits extended into the phylum Basidiomycota. Multiple sequence alignment led to the identification of a network of seven conserved tryptophans that surround the β‐lactamase‐like region. This is the first report of a predicted β‐lactamase that is an endoprotease.

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.