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No description available.

No description available.

This dissertation describes the characterization of a novel transposable element isolated from the plant pathogenic fungus Magnaporthe grisea. The sequence of MGR583, a previously reported repeated DNA fragment, was completed and shown to have features characteristic of non-LTR retroelements (LINEs). These include an element length of 5.9 kb, the lack of flanking long terminal repeats, the presence of short (6-13 bp) direct repeats flanking many element copies, and two principal open reading frames (ORFs). The first ORF is 570 amino acids in length and contains homology to the gag ORFs found in many retroelements. The second ORF is 1,295 amino acids in length and has strong homology to reverse transcriptases (RT) ORFs found in non-LTR retroelements (LINEs). In accordance with these results, the name of the repeat was changed to MGL for Magnaporthe grisea LINE. Analysis of the 3' terminus of MGL showed 90% homology to the 3' terminus of Mg-SINE, suggesting an evolutionary relationship between these two elements. A survey of the distribution of MGL in populations of M. grisea showed the element to be present in all isolates tested. Copy number was not uniform between isolates, with approximately fifty copies present in rice isolates and between less than 10 and up to 50 copies in the 17 non-rice isolates tested. A PCR-based assay was designed and used to screen M. grisea isolates for polymorphic MGL insertion loci. Thirteen polymorphic MGL insertions were scored and used to construct a phylogenetic tree that included 11 non-rice isolates and 20 rice isolates. The results strongly suggested that development of virulence on rice was a single event correlated with the acquisition of virulence on several other grass species. In addition, the observation that rearrangements occurred at one of the insertion loci in some rice isolate strains support the proposal that there is considerable plasticity in the genomes of these isolates. Finally, a yeast transposon ( Tyl) system was used to express and test the second ORF for RT activity. No activity was detected for any of the MGL RT constructs tested.

The biological control bacterium Pseudomonas aureofaciens strain 30-84 produces three phenazine antibiotics. Phenazines are responsible for pathogen inhibition by strain 30-84 as well as its ability to persist in the rhizosphere. Although this bacterium can suppress take-all of wheat disease when applied as a seed inoculum, performance of this agent, as with many biological control agents, can be variable in the field. A factor in establishment and pathogen inhibition may be the indigenous microbial community that competes with strain 30-84 and may interfere with phenazine production as a competitive mechanism. In this study, a wheat rhizosphere microbial community library was screened and ca. 4% of the isolates were found to inhibit phenazine production by strain 30-84. A sub-group of these isolates was characterized and found to produce extracellular signals that suppressed phenazine gene expression. The signal from isolate PU-15 was initially characterized and appeared to be chemically and mechanistically unlike other known negative-acting signals. A genetic region was cloned from this isolate that decreased phenazine gene expression and production in strain 30-84. Negative communication also affected the ability of strain 30-84 to inhibit the pathogenic fungus Gaeuman-nomyces graminis pv. tritici in vitro. Therefore, negative communication may contribute to the inconsistencies of biological control in the field.

The fungus Nectria haematococca is a broad host range pathogen. Isolates pathogenic on pea are able to detoxify the phytoalexin pisatin using the enzyme pisatin demethylase. When the gene (PDA1 ) encoding this enzyme was mutated via gene disruption, the mutants were less virulent but still pathogenic on pea. Additional studies demonstrated that PDA1 was on a 1.6-Mb conditionally dispensable (CD) chromosome and that loss of this CD chromosome resulted in the complete loss of pea pathogenicity. This leads to the hypothesis that there are other pea p̱athogenicity (PEP) genes in addition to the PDA1 gene on the CD chromosome. One of the major goals of this work was to test this hypothesis by isolating and characterizing these PEP genes. The results identified three novel PEP genes: PEP1, PEP2, and PEP5, each of which can confer disease-causing ability independently when introduced into a nonpathogenic isolate lacking the CD chromosome. The predicted product of PEP5 is related to members of the major facilitator superfamily, including proton-dependent multidrug export systems, and the predicted product of PEP2 contains conserved RNA-binding motifs. PEP1 shows no significant similarity to any known gene in the public databases. The three PEP genes and PDA1 are organized into a functional cluster, termed the PEP cluster, within 25 kb that conditions full pathogenicity on pea. The PEP cluster contains two additional genes, cDNA3 and cDNA4, and neither gene by itself is able to confer disease-causing abilities. The open reading frame (ORF) for cDNA3 gene is small. The predicted cDNA4 product exhibits significant similarities to the transposon impala of Fusarium oxysporum. The sequences of other portions of the PEP cluster predict four ORFs showing strong similarities to other fungal transposases. Several features of the PEP cluster, such as possession of multiple virulence genes, presence of DNA mobile elements, and differences in both codon usage and G+C content compared with other portions of the genome, resemble those of the pathogenicity islands identified in plant and animal bacterial pathogens. These properties raise the possibility that the PEP gene cluster may represent a fungal pathogenicity island. The second major goal of my work was to quantify the expression of PDA1, PEP1, PEP2, and PEP5 in vitro and in planta, and to characterize the regions flanking the PEP cluster. A real-time quantitative RT-PCR approach was used to measure the mRNA levels of these pathogenicity genes. In a glucose-based growth medium, mRNA levels of PDA1, PEP1, PEP5 were very low, while expression of PEP2 was undetectable. Starvation in vitro strongly stimulated PDA1 gene expression, whereas expression of PEP1 and PEP5 increased only moderately. In contrast, starvation had no effect on expression of PEP2 as indicated by an undetectable mRNA level during the 12 hr time course tested. In vitro pisatin strongly induced the expression of all four pathogenicity genes and the PDA1 experienced the highest level of induction (∼300-fold increase). Finally, marked induction of PDA1, PEP1 and PEP2 was observed during infection of pea roots, whereas the expression of PEP5 was only moderately induced. The flanking regions of the PEP cluster were sequenced and the sequences predict six ORFs that display significant similarities to various fungal genes. The G+C content, gene density, and codon preference of the PEP cluster and its flanking regions are similar. All six of the predicted genes in the flanking regions of the PEP cluster are expressed during infection of pea.

Magnaporthe grisea is a filamentous ascomycete fungus that causes blast disease on rice and other grasses. Blast is a serious deterrent to rice production and negatively affects production of other cereals, forage crops and economically important grasses. The primary means of blast disease management involves the development and implementation of genetically resistant plants. Understanding the molecular basis of plant resistance is the foundation for the development of unique and durable plant protection. The results presented here focus on genes in the rice blast fungus called avirulence genes that encode molecules acting as effectors of host resistance. Until recently, two avirulence loci had been shown to induce resistance in rice cultivar Maratelli. This study gives an update on the current status of one, the AVR1-MARA locus, and describes a new Maratelli avirulence locus that is not allelic to AVR1-MARA or AVR2-MARA. Additionally, evidence is given that indicates a genome rearrangement is responsible for generation of the newly described avirulence locus. Genetic data, hybridization results and DNA sequence analysis demonstrate the translocation of a large AT-rich fragment from one chromosomal location to another. Molecular detection of the translocation is demonstrated by hybridization of certain AVR1-MARA markers that only follow the avirulent phenotype in strains after the rearrangement. The rearrangement is detectable genetically, as the avirulent phenotype controlled at this locus segregates independently from progenitor strains that also contain a single Maratelli-specific avirulence gene. CHEF electrophoretic separation of chromosome-sized DNA shows that the AT rich sequences are located on one of the larger M. grisea chromosomes both before and after cross 4134. Hybridization of CHEF blots indicates that two chromosomes may have been involved in a translocation, however a reorganization of chromosome 2 cannot be ruled out. A homing enzyme strategy for determining the size of the translocated fragment is described. These results demonstrate an example of genomic plasticity leading to a translocation and creation of a new avirulence locus in the rice blast fungus M. grisea.

α-Tomatine, synthesized by Lycopersicon species, is toxic to a broad range of fungi and has long been considered a potential barrier to microbial infection. I hypothesize that a successful tomato pathogen must overcome this toxic compound in order to parasitize the plant. In this study, I have examined a number of saprophytes, non-pathogens of tomato, and tomato pathogens for their tolerance to α-tomatine and their ability to enzymatically degrade this compound. Although, overall, fungal saprophytes and non-pathogens of tomato were sensitive to α-tomatine while tomato pathogens were very tolerant, several tomato pathogens were quite sensitive. All tomato pathogens, except the Pythiaceous fungi, were able to degrade α-tomatine to the less toxic compounds β₂-tomatine or tomatidine. Many saprophytes and non-pathogens were sensitive to these degradation products while tomato pathogens were tolerant suggesting that tomato pathogens may have also acquired a non-degradative means to tolerate α-tomatine and its derivatives. As an initial step to evaluate the importance of detoxification of α-tomatine, I have purified to homogeneity a β-1,2-D glucosidase from the tomato leaf pathogen Septoria lycopersici that hydrolyzes the β-1,2-D glucosyl bond on the tetrasaccharide moiety of α-tomatine to produce β₂-tomatine and glucose. Little to no enzyme activity was detected when other β-1,2-D glycosides were used as substrates suggesting that this organism has evolved a special means to overcome α-tomatine. The gene encoding this protein, called β₂-tomatinase, was isolated from S. lycopersici and a β₂-tomatinase homologue from the green fruit pathogen Colletotrichum coccodes. The β₂-tomatinase genes in C. coccodes and S. lycopersici were mutated via transformation-mediated gene disruption and the β₂-tomatinase mutants were analyzed for their tolerance to α-tomatine and their ability to parasitize tomato. The S. lycopersici β₂-tomatinase mutant became 12 times more sensitive to α-tomatine. The C. coccodes β₂-tomatinase mutant still retained its tolerance to α-tomatine, its ability to degrade α-tomatine to the aglycone tomatidine, and its ability to parasitize the tomato fruit but was deficient in β₂-tomatinase activity. Expression of the S. lycopersici β₂-tomatinase gene in N. haematocacca, a pea pathogen, increased its tolerance to α-tomatine and these transformants acquired the ability to form lesions on green tomato fruit, an organ containing α-tomatine.

Root microbe interactions are initiated generally in the region of elongation while the root tip remains infection free. In this dissertation, the hypothesis that root border cells play a role in protecting the root tip from fungal infections was examined. When radicles of pea seedlings were inoculated with Nectria haematococca , the zone of elongation became infected in the majority of the samples (ca. 90%) but the root tips appeared infection free and continued to grow essentially normally. However, microscopic evaluation revealed that N. haematococca propagules germinated and colonized border cells, leading to the formation of "mantles" that ensheathed the root tip. The tip tissue, nevertheless, remained infection free. Removing border cells prior to inoculation with the fungus or retarding their separation increased tip infections. Additionally, exudates from fungus inoculated root tips inhibited fungal growth in vitro when compared to those from uninoculated seedlings. When root tip infection occurred, a strong correlation between tip infection and cessation of further root growth was observed. In response to root tip infection by N. haematococca, a concomitant induction of border cell separation and defense gene expression in the tip was observed, suggesting that border cell separation may be a previously unrecognized alternative defense strategy of the pea root tip. The results are consistent with the following model: The separation of border cells, together with the presence of an inhibitory compound in the root exudates, minimizes contact between the fungus and the root tip thus protecting the tip from infection. Under conditions when tip infection does occur, the active induction of renewed border cell separation serves to remove infected tissue from the apical meristem area. Plant mutants altered in border cell separation can be used in the future studies to definitively establish the role of border cells in plant health.

Many plants produce toxic compounds, called phytoalexins, in response to infection by microorganisms. Some fungal pathogens of these plants can detoxify their host's phytoalexins and genetic studies of the ascomycete, Nectria haematococca Mating Population VI have established an association between detoxification of the pea phytoalexin, pisatin (Pda), and pathogenicity. Previous studies of one of the six genes (PDA) that confer this trait (PDA6-1) was on a dispensable chromosome. In the current study, a technique was developed that uses the pea plant to select for highly virulent recombinant progeny from crosses in which such progeny were relatively rare. It was demonstrated that when pea plants are inoculated with a mixture of ascospores that isolates recovered from pea lesions showed a strong bias for Pda and for being more virulent on pea, compared with ascospore progeny which did not undergo selection on plant. Additionally, all highly virulent isolates had PDA1-1, one of the three PDA genes present in the cross parents, showing that PDA1-1, or a linked gene, was necessary for virulence on pea. In the current study, highly virulent isolates were also identified by screening progeny from crosses that involved a highly virulent parent, 34-18. Analysis of 34-18 and its progeny showed that this isolate contains three PDA genes, PDA5 and PDA9, which were characterized in this study, and an allele of a previously characterized PDA gene. All three genes were associated with virulence on pea and could be lost during genetic crosses. Electrophoretic karyotype (CHEF) analysis showed that this was due to loss of a 1.5 Megabase chromosome carrying PDA1-2 and at least a portion of a 4.9 Mb chromosome carrying PDA5 and PDA9. CHEF analysis also showed that the other previously characterized PDA genes (PDA1-1, PDA2, PDA3, PDA4, PDA6-1 and PDA6-2) were on dispensable chromosomes. These dispensable chromosomes were not required for pathogenicity on carrot and ripe tomato. The results from this work provide evidence to support the hypothesis that the PDA chromosomes are dispensable, that some of them contain genes conferring virulence specifically on pea and genes for pathogenicity on other hosts were on non-PDA chromosomes.