Search

Plants produce low molecular weight compounds with antimicrobial activity in response to microbial attack termed phytoalexins. The first phytoalexin identified was (+) pisatin from pea, and several fungi are able to detoxify pisatin to a less inhibitory compound, including F. oxysporum f. sp. pisi. This detoxification is catalyzed by demethylation of the compound (termed pisatin demethylase activity, or PDA) by the cytochrome P450, Pda. Here we detail two procedures to assess PDA using radiolabeled [

Plants produce low molecular weight compounds with antimicrobial activity in response to microbial attack termed phytoalexins. The first phytoalexin identified was (+) pisatin from pea, and several fungi are able to detoxify pisatin to a less inhibitory compound, including F. oxysporum f. sp. pisi. This detoxification is catalyzed by demethylation of the compound (termed pisatin demethylase activity, or PDA) by the cytochrome P450, Pda. Here we detail two procedures to assess PDA using radiolabeled [14C]pisatin as a substrate and monitoring activity using a scintillation counter.

The pea pathogen Fusarium oxysporum f. sp. pisi is able to detoxify pisatin produced as a defense response by pea, and the gene encoding this detoxification mechanism, FoPDA1, was 82% identical to the cytochrome P450 pisatin demethylase PDA1 gene in Nectria haematococca. A survey of F. oxysporum f. sp. pisi isolates demonstrated that, as in N. haematococca, the PDA gene of F. oxysporum f. sp. pisi is generally located on a small chromosome. In N. haematococca, PDA1 is in a cluster of pea pathogenicity (PEP) genes. Homologs of these PEP genes also were found in the F. oxysporum f. sp. pisi isolates, and PEP1 and PEP5 were sometimes located on the same small chromosomes as the FoPDA1 homologs. Transforming FoPDA1 into a pda– F. oxysporum f. sp. lini isolate conferred pda activity and promoted pathogenicity on pea to some transformants. Different hybridization patterns of FoPDA1 were found in F. oxysporum f. sp. pisi but these did not correlate with the races of the fungus, suggesting that races within this forma specialis arose independently of FoPDA1. FoPDA1 also was present in the formae speciales lini, glycines, and dianthi of F. oxysporum but they had mutations resulting in nonfunctional proteins. However, an active FoPDA1 was present in F. oxysporum f. sp. phaseoli and it was virulent on pea. Despite their evolutionary distance, the amino acid sequences of FoPDA1 of F. oxysporum f. sp. pisi and F. oxysporum f. sp. phaseoli revealed only six amino acid differences, consistent with a horizontal gene transfer event accounting for the origin of these genes.

Many secondary metabolites that are normally undetectable or in low amounts in healthy plant tissue are synthesized in high amounts in response to microbial infection. Various abiotic and biotic agents have been shown to mimic microorganisms and act as elicitors of the synthesis of these plant compounds. In the present study, sub-lethal levels of electric current are shown to elicit the biosynthesis of secondary metabolites in transgenic and non-transgenic plant tissue. The production of the phytoalexin (+)-pisatin by pea was used as the main model system. Non-transgenic pea hairy roots treated with 30-100 mA of electric current produced 13 times higher amounts of (+)-pisatin than did the non-elicited controls. Electrically elicited transgenic pea hairy root cultures blocked at various enzymatic steps in the (+)-pisatin biosynthetic pathway also accumulated intermediates preceding the blocked enzymatic step. Secondary metabolites not usually produced by pea accumulated in some of the transgenic root cultures after electric elicitation due to the diversion of the intermediates into new pathways. The amount of pisatin in the medium bathing the roots of electro-elicited roots of hydroponically cultivated pea plants was 10 times higher 24 h after elicitation than in the medium surrounding the roots of non-elicited control plants, showing not only that the electric current elicited (+)-pisatin biosynthesis but also that the (+)-pisatin was released from the roots. Seedlings, intact roots or cell suspension cultures of fenugreek (Trigonella foenum-graecum), barrel medic, (Medicago truncatula), Arabidopsis thaliana, red clover (Trifolium pratense) and chickpea (Cicer arietinum) also produced increased levels of secondary metabolites in response to electro-elicitation. On the basis of our results, electric current would appear to be a general elicitor of plant secondary metabolites and to have potential for application in both basic and commercial research.

S-adenosyl-l-methionine: 2-hydroxyisoflavanone 4'-O-methyltransferase (HI4'OMT) methylates 2,7, 4'-trihydroxyisoflavanone to produce formononetin, an essential intermediate in the synthesis of isoflavonoids with methoxy or methylenedioxy groups at carbon 4' (isoflavone numbering). HI4'OMT is highly similar (83% amino acid identity) to (+)-6a-hydroxymaackiain 3-O-methyltransferase (HMM), which catalyzes the last step of (+)-pisatin biosynthesis in pea. Pea contains two linked copies of HMM with 96% amino acid identity. In this report, the catalytic activities of the licorice HI4'OMT protein and of extracts of Escherichia coli containing the pea HMM1 or HMM2 protein are compared on 2,7,4'-trihydroxyisoflavanone and enantiomers of 6a-hydroxymaackiain. All these enzymes produced radiolabelled 2,7-dihydroxy-4'-methoxyisoflavanone or (+)-pisatin from 2,7,4'-trihydroxyisoflavanone or (+)-6a-hydroxymaakiain when incubated with [methyl-(14)C]-S-adenosyl-l-methionine. No product was detected when (-)-6a-hydroxymaackiain was used as the substrate. HI4'OMT and HMM1 showed efficiencies (relative V(max)/K(m)) for the methylation of 2,7,4'-trihydroxyisoflavanone 20 and 4 times higher than for the methylation of (+)-6a-hydroxymaackiain, respectively. In contrast, HMM2 had a higher V(max) and lower K(m) on (+)-6a-hydroxymaackiain, and had a 67-fold higher efficiency for the methylation of (+)-6a-hydroxymaackiain than that for 2,7,4'-trihydroxyisoflavanone. Among the 15 sites at which HMM1 and HMM2 have different amino acid residues, 11 of the residues in HMM1 are the same as found in HI4'OMTs from three plant species. Modeling of the HMM proteins identified three or four putative active site residues responsible for their different substrate preferences. It is proposed that HMM1 is the pea HI4'OMT and that HMM2 evolved by the duplication of a gene encoding a general biosynthetic enzyme (HI4'OMT).

Most plants synthesize antimicrobial compounds as part of normal plant development (i.e., phytoanticipins) or synthesize such compounds de novo when challenged by microorganisms (i.e., phytoalexins). The presumed role of these plant antibiotics is to protect the plant from disease. However, many phytopathogenic fungi have enzymes that can detoxify the phytoanticipins or phytoalexins produced by their host. This may be a means that these pathogens have evolved to circumvent resistance mechanisms based on the production of plant antibiotics. Many of the phytoanticipin- and phytoalexin-detoxifying enzymes produced by phytopathogenic fungi have biochemical and regulatory properties that would indicate the phytoanticipins and phytoalexins produced by their host are their normal substrates. In addition, their activity, enzymatic products, or transcripts can be detected in infected plant tissue suggesting that they are functioning in planta during pathogenesis. Specific mutations have been made by transformation-mediated gene-disruption procedures that eliminate the ability of Gaeumannomyces graminis var. avenae, Gloeocercospora sorghi, and Nectria haematococca to detoxify the phytoanticipins or phytoalexins produced by their hosts. The effect of these mutations on pathogenicity indicates a requirement for detoxifying enzymes in G. graminis var. avenae but not in G. sorghi or N. haematococca. Key words: disease resistance, pathogenicity mechanisms, isoflavonoids, saponins, cyanide.

The ascomycetous fungus Nectria haematococca, (asexual name Fusarium solani), is a member of a group of >50 species known as the “Fusarium solani species complex”. Members of this complex have diverse biological properties including the ability to cause disease on >100 genera of plants and opportunistic infections in humans. The current research analyzed the most extensively studied member of this complex, N. haematococca mating population VI (MPVI). Several genes controlling the ability of individual isolates of this species to colonize specific habitats are located on supernumerary chromosomes. Optical mapping revealed that the sequenced isolate has 17 chromosomes ranging from 530 kb to 6.52 Mb and that the physical size of the genome, 54.43 Mb, and the number of predicted genes, 15,707, are among the largest reported for ascomycetes. Two classes of genes have contributed to gene expansion: specific genes that are not found in other fungi including its closest sequenced relative, Fusarium graminearum; and genes that commonly occur as single copies in other fungi but are present as multiple copies in N. haematococca MPVI. Some of these additional genes appear to have resulted from gene duplication events, while others may have been acquired through horizontal gene transfer. The supernumerary nature of three chromosomes, 14, 15, and 17, was confirmed by their absence in pulsed field gel electrophoresis experiments of some isolates and by demonstrating that these isolates lacked chromosome-specific sequences found on the ends of these chromosomes. These supernumerary chromosomes contain more repeat sequences, are enriched in unique and duplicated genes, and have a lower G+C content in comparison to the other chromosomes. Although the origin(s) of the extra genes and the supernumerary chromosomes is not known, the gene expansion and its large genome size are consistent with this species' diverse range of habitats. Furthermore, the presence of unique genes on supernumerary chromosomes might account for individual isolates having different environmental niches., Author Summary Nectria haematococca MPVI occurs as a saprophyte in diverse habitats and as a plant and animal pathogen. It also was the first fungus shown to contain supernumerary chromosomes with unique habitat-defining genes. The current study reveals that it has one of the largest fungal genomes (15,707 genes), which may be related to its habitat diversity, and describes two additional supernumerary chromosomes. Two classes of genes were identified that have contributed to gene expansion: 1) specific genes that are not found in other fungi, and 2) genes that are present as multiple copies in N. haematococca but commonly occur as a single copy in other fungi. Some of these genes have properties suggesting their acquisition by horizontal gene transfer. We show that the three supernumerary chromosomes are different from the normal chromosomes; they contain more repeat sequences, are particularly enriched in unique and duplicated genes, and have a lower G+C content. Additionally, the biochemical functions of genes on these chromosomes suggest they may be involved in niche adaptation. The dispensable nature and possession of habitat-determining genes by these chromosomes make them the biological equivalent of bacterial plasmids. We believe they contribute to microbial diversity and have been overlooked in models of fungal evolution.

To explore the effectiveness of insect derived protease inhibitors in protecting plants against insect feeding, anti-trypsin, anti-chymotrypsin and anti-elastase protease inhibitor (PI) genes from Manduca sexta L. were expressed in transgenic cotton (Gossypium hirsutum L.). From 198 independent transformants, 35 elite lines were further analyzed. Under the control of the 35S promoter of CaMV, PI accumulated to approximately 0.1% of total protein, depending on the tissue analyzed. Using cell-flow cytometry, DNA content/ nuclei of transgenic and non-transformed cotton were identical. On cotton plants expressing PIs, fecundity of Bemisia tabaci (Genn.), the sweetpotato whitefly, was reduced compared to controls. Expression of these protease inhibitors may reduce the developmental rate of B. tabaci and other insects, and provide a strategy for cotton protection.

Nectria haematococca MPVI can be found in many different biological habitats but has been most studied as a pathogen of pea (Pisum sativum). Genetic analyses of isolates obtained from a variety of biological sources has indicated that a number of genes control pathogenicity on pea but that one important PEa Pathogenicity (PEP) gene is PDA, which confers the ability to detoxify the pea phytoalexin pisatin. In these studies, all naturally occurring isolates that lacked PDA (i.e. Pda- isolates) and all Pda- progeny were essentially non-pathogenic on pea. However, we have demonstrated recently that Pda- mutants created by transformation-mediated gene disruptions, while having a modest reduction in virulence, and more virulent than any naturally occurring Pda- isolates. In addition we know that PDA genes are on dispensable (DS) chromosomes in this fungus. We believed that the gene disruption mutants have allowed the detection of other PEP genes that are present on the DS chromosomes along with PDA and that naturally occurring Pda- isolates usually lack this DS chromosome. This would explain why naturally occurring Pda- isolates are always low in virulence. We propose that the DS chromosomes in fungi are analogous to bacterial plasmids which allow those microorganisms to colonise different habitats, i.e. the DS chromosomes of Nectria haematococca contain genes that allow individual isolates of this broad host range pathogen to occupy different biological niches.

This paper focuses on research reported since the 1992 ISMPMI meeting that has attempted to identify pathogenicity genes in fungi. One category we discuss consists of those genes which encode known biochemical traits that historically have been “logical” candidates for functioning in pathogenicity. In addition, we present a summary of the results of some of the newer approaches to isolate pathogenicity genes that are independent of prior knowledge of the activities encoded by the gene. In this paper we do not consider genes whose absence is required for pathogenicity (ie. avirulence genes or genes which induce a non-specific hypersensitive response).

As one approach to alleviating the need for insecticide spraying, our objective is to express protein insecticides in transgenic alfalfa. To initiate these studies, a cDNA encoding the protease inhibitor (PI) anti-elastase from Manduca sexta was placed under the control of the CaMV 35S promoter, inserted into pAN 70, and transferred into leaf and petiole sections of alfalfa (Medicago sativa L.) using Agrobacterium tumefaciens mediated gene transfer. Transformation rates were 10% of all explants exposed to Agrobacterium. More than 1000 transgenic plants containing the PI have been recovered. Transgenic plants were initially identified when leaf explants from the regenerated plants formed callus in the presence of 50 μg/ml kanamycin, and subsequently the presence of the PI gene was confirmed by southern analysis. The 35S promoter-PI fusion produced up to 0.125% of total protein as PI protein in leaves, roots, and flowers. Progeny analysis demonstrated Mendelian segregation of the NPTII gene (observed as kanamycin resistance) and the PI (confirmed by southern analysis). Accumulation of the anti-elastase PI insecticide in transgenic alfalfa reduced the onset of thrip predation, suggesting that this methodology can establish insect resistance within this agronomically important legume.

We compared the transport in vitro of fusion proteins of neomycin phosphotransferase II (NPTII) with either the transit peptide of the small subunit (SSU) of ribulose-1,5-bisphosphate carboxylase/oxygenase or the transit peptide and the 23 aminoterminal amino acids of the mature small subunit. The results showed that the transit peptide is sufficient for import of NPTII. However, transport of the fusion protein consisting of the transit peptide linked directly to NPTII was very inefficient. In contrast, the fusion protein containing a part of the mature SSU was imported with an efficiency comparable to that of the authentic SSU precursor. We conclude from these results that other features of the precursor protein in addition to the transit peptide are important for transport into chloroplasts. In order to identify functional regions in the transit peptide, we analyzed the transport of mutant fusion proteins. We found that the transport of fusion proteins with large deletions in the aminoterminal, or central part was drastically reduced. In contrast, duplication of a part of the transit peptide led to a marked increase in transport.

Import of the small subunit of ribulose-1,5-biphosphate carboxylase/oxygenase into the chloroplast has been proposed to involve two proteolytic cleavages which convert the 20-kDa precursor (pSSU) into the mature 14-kDa subunit (SSU) via an 18-kDa intermediate. A deletion mutant (PSd48/57) of pSSU which lacks 10 amino acids in a conserved region in the carboxyl-terminal portion of the transit peptide is converted into a series of 16-18-kDa polypeptides in addition to the mature 14-kDa SSU when imported into isolated pea chloroplasts. We examined import and processing of this mutant pSSU to determine whether the 16-18-kDa SSUs undergo further maturation in the chloroplast stroma to yield 14-kDa SSU. The ratio of incorrectly processed to 14-kDa SSU is stable up to 60 min following import. This indicates that processing of PSd48/57 involves a single proteolytic cleavage which occurs during or immediately following transit across the chloroplast envelope. The carboxyl-terminal portion of the transit peptide confers either sequence specificity for the processing protease or provides a three-dimensional structure necessary for consistent cleavage at the mature amino terminus of SSU. Incorrectly processed SSUs were incorporated into the holoenzyme demonstrating that removal of the entire transit sequence is not necessary for assembly of the holoenzyme.

We studied transport and binding to intact chloroplasts of 10 mutants in three regions of the transit peptide of a precursor to the small subunit of ribulose 1,5-bisphosphate carboxylase/oxygenase [3-phospho-D-glycerate carboxy-lyase (transphosphorylating), E.C.4.1.1.39]. Transport was assayed in a reconstituted system using isolated pea chloroplasts and radioactively labeled precursor. Binding to the chloroplast envelope was assayed in a similar manner using chloroplasts pretreated with nigericin. Most mutants showed a dramatically decreased capacity of binding, although some of them transported relatively well. The accumulation of the mutant proteins inside the chloroplast as a function of time was examined. Although the authentic small subunit precursor was imported rapidly, uptake of most mutant precursors was considerably slower and continued until the last time point examined. In terms of assigning functions to individual regions, we found that at least the middle region and parts of the amino and the carboxyl termini of the transit peptide are more important for receptor binding than for translocation. A two-step processing mechanism has been postulated for the maturation of the small subunit precursor. This model predicts the occurrence of processing intermediates. When precursors carrying carboxyl-terminal deletions were presented to the chloroplast, no defined intermediates could be detected. Instead, a number of proteins, probably resulting from aberrant processing, accumulated simultaneously inside the chloroplasts.

Deletion mutations, 3-19 amino acids in size, were introduced into the transit peptide (57 amino acids) of a small subunit (SSU) of ribulose-1,5-bisphosphate carboxylase/oxygenase from pea. Transport of the authentic small subunit precursor (pSSU) and of the mutant pSSUs by isolated chloroplasts of pea was examined. We show that the transit peptide contains two different, separated functional regions. A deletion mutation in the central region of the transit peptide, a region purported to be important for function, barely affected transport. Changes in the amino-terminal region of the transit peptide appeared normal. A deletion mutation at the carboxy-terminus of the transit peptide interfered with both transport and processing. From the aberrant processing we suggest that pSSU is matured in more than one step, and that the maturation signal is located within the carboxy-terminal 16 amino acids. The methionine residue at the evolutionarily conserved cleavage site (cysteine-methionine) between the transit peptide and the mature protein is not essential processing.

Chloroplast import and processing of two precursor proteins with mutations in the carboxyl-terminal region of the transit peptide were examined in vitro. Deletion mutations were introduced into the 57-amino acid transit peptide of a chloroplast protein, the small subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase, from pea. A mutant, PSd48/57, in which nine carboxyl-terminal amino acids of the transit peptide had been deleted, was imported and processed to a series of 13- to 18-kDa polypeptides including the 14-kDa mature small subunit. In contrast, processing of a mutant, PSd45/57, in which an additional three amino acids had been removed, resulted in a series of polypeptides which did not include the mature small subunit. Whereas PSd48/57 was imported as efficiently as the wild-type precursor, import of PSd45/57 was only 25% as efficient as that of the authentic precursor. The mutant precursor proteins PSd48/57 and PSd45/57 are distinguished by a three-amino acid sequence, Ile-Thr-Ser, located in the carboxyl-terminal region of the transit peptide. We show that all or part of this sequence is required for correct processing.

The mature small subunit (SSU) of ribulose-1,5-bisphosphate carboxylase/oxygenase (EC 4.1.1.39) in higher plants contains a highly conserved sequence of 16 amino acids that is absent in the SSUs of cyanobacteria. To determine whether this region of the SSU of higher plants has a specific function, portions of the SSU genes (rbcS) of pea (Pisum sativum) and the cyanobacterium Anacystis nidulans were fused to create chimeric genes that either lacked or contained the coding sequence for the 16 conserved amino acids. Precursor proteins synthesized in vitro from the chimeric genes were incubated with isolated pea chloroplasts to assay import and assembly into the holoenzyme. Fusion proteins lacking the 16-amino acid sequence were imported and processed but failed to assemble with endogenous large subunit. Addition of the region from a pea rbcS containing the 16 amino acids to the rbcS of Anacystis enabled the imported SSU fusion protein to assemble with pea large subunit. This 16-amino acid sequence is encoded by a separate exon in certain rbcS genes of higher plants. We propose that the conserved 16-amino acid sequence constitutes a domain acquired to facilitate assembly of the eukaryotic holoenzyme.