FvSTUA is a Key Regulator of Sporulation, Toxin Synthesis, and Virulence in Fusarium verticillioides.

Fusarium verticillioides is one of the most important pathogens of maize, causing rot and producing fumonisin mycotoxins during infection. Ingestion of fumonisin-contaminated corn causes underperformance and even fatal toxicity in livestock and is associated with neural tube birth defects, growth stunting in children, and some cancers. StuA, an APSES-class transcription factor, is a major developmental transcriptional regulator in fungi. It has been shown to regulate crucial developmental processes, such as sporulation, virulence, and mycotoxin synthesis among others. In this study, the role of FvSTUA in F. verticillioides was examined by characterizing ∆FvstuA deletion mutants functionally and transcriptomally. The deletion mutants exhibited reduced vegetative growth, stunted aerial hyphae, and significant reductions in microconidiation. Macroconidiation and hydrophobicity of the deletion strains were reduced as well. Additionally, fumonisin production and virulence of the deletion mutants were greatly reduced. Transcriptomic analysis revealed downregulation of expression of several genes in the fumonisin and fusarin C biosynthetic clusters and differential expression of genes involved in conidiation and virulence. Nuclear localization of FvSTUA supported its likely function as a transcription factor. Together, our results indicate that FvSTUA plays a global role in transcriptional regulation in F. verticillioides influencing morphogenesis, toxin production, and virulence.

Bacillus mojavensis RRC101 Lipopeptides Provoke Physiological and Metabolic Changes During Antagonism Against Fusarium verticilliodes.

The mycotoxigenic pathogen Fusarium verticillioides threatens the quality and utility of maize across industrial and agricultural purposes. Chemical control is complicated by the intimate endophytic lifestyle of the pathogen with its host. Bacillus mojavensis RRC101, a maize-endophytic bacterium, has been observed to reduce F. verticillioides disease severity and fumonisin accumulation when coinoculated to maize. Genome sequencing and annotation identified a number of biocontrol-relevant pathways in RRC101. Biochemical assays confirmed the presence and activity of surfactin- and fengycin-type lipopeptides, with fengycins responsible for antifungal activity against F. verticillioides. This antagonism manifests as inhibition of filamentous growth, with microscopy revealing hyphal distortions, vacuolization, and lysis. F. verticillioides secondary metabolism also responds to antagonism, with lipopeptide challenge inducing greater fumonisin production and, in the case of fengycins, eliciting pigment accumulation at sites of inhibition. Together, these data suggest that antibiotic and toxin production are components of a complex biochemical interaction among maize endophytes, one pathogenic and one beneficial.

Plant pathogen forensics: capabilities, needs, and recommendations.

A biological attack on U.S. crops, rangelands, or forests could reduce yield and quality, erode consumer confidence, affect economic health and the environment, and possibly impact human nutrition and international relations. Preparedness for a crop bioterror event requires a strong national security plan that includes steps for microbial forensics and criminal attribution. However, U.S. crop producers, consultants, and agricultural scientists have traditionally focused primarily on strategies for prevention and management of diseases introduced naturally or unintentionally rather than on responding appropriately to an intentional pathogen introduction. We assess currently available information, technologies, and resources that were developed originally to ensure plant health but also could be utilized for postintroduction plant pathogen forensics. Recommendations for prioritization of efforts and resource expenditures needed to enhance our plant pathogen forensics capabilities are presented.

Distinct roles of CONSTANS target genes in reproductive development of Arabidopsis.

In plants, flowering is triggered by endogenous and environmental signals. CONSTANS (CO) promotes flowering of Arabidopsis in response to day length. Four early target genes of CO were identified using a steroid-inducible version of the protein. Two of these genes, SUPPRESSOR OF OVEREXPRESSION OF CO 1 (SOC1) and FLOWERING LOCUS T (FT), are required for CO to promote flowering; the others are involved in proline or ethylene biosynthesis. The SOC1 and FT genes are also regulated by a second flowering-time pathway that acts independently of CO. Thus, early target genes of CO define common components of distinct flowering-time pathways.

Volatiles produced by Bacillus mojavensis RRC101 act as plant growth modulators and are strongly culture-dependent.

Volatile organic compounds (VOCs) produced by Plant Growth Promoting Rhizobacteria have recently been investigated due to their role in plant growth promotion and defense. Whereas some bacterial VOCs like 3-hydroxy-2-butanone (acetoin) and 2,3-butanediol produced by strains of Bacillus subtilis and Bacillus amyloliquefaciens promote plant growth, others like hydrogen cyanide and 3-phenylpropionic acid are phytotoxic, inhibiting plant growth. Bacillus mojavensis, a close relative of B. subtilis, is an endophytic bacterium of maize that has been shown to have antagonistic activity against the mycotoxigenic phytopathogen Fusarium verticillioides and growth promotion activity on maize seedlings. To investigate the growth promotion activity of B. mojavensis, Arabidopsis thaliana seedlings were grown on 1/2x Murashige & Skoog (MS) medium in divided Petri dishes while bacteria were grown either on 1/2x MS or nutrient agar (NA) medium, so that only microbial volatiles reached the seedlings. Significant plant growth promotion in Arabidopsis seedlings was observed when 1/2x MS medium was used for bacterial growth. In contrast, phytotoxicity was observed with bacterial growth on NA medium. These results indicate that VOCs produced by B. mojavensis may act as plant growth modulators rather than just promoters. Using Solid Phase Microextraction (SPME) coupled with GC-MS, the plant growth promoting compounds acetoin and 2,3-butanediol were both identified as being produced by B. mojavensis on growth promoting 1/2x MS medium. In contrast, while no phytotoxic VOC was conclusively identified from B. mojavensis on NA medium, detection of relatively high levels of acetone/2-propanone indicates its possible contribution to Arabidopsis phytotoxicity.

New (and used) approaches to the study of fungal pathogenicity.

The fungi are the most economically important plant pathogens and continue to be the focus of extensive research with a wide variety of methodologies. Enhancements in microscopy techniques have increased our ability to visualize the intimate interaction of fungi and their host plants. Improving methods allow pharmacological inhibition and genetic dissection of the determinants of fungal pathogenicity in a gene-by-gene approach. Identification and analysis of genes differentially transcribed in ways pertinent to pathogenicity continues to be a frequent research approach. Genome-wide analysis is gaining favor in biological research and fungal plant pathogens are no exception. Several industrial research groups are exploring fungal plant pathogenesis based on genomic sequence data and genome-wide mutagenesis. In March 2001 the first publicly available complete genome of a filamentous fungus (Neurospora crassa) was released. N. crassa is of course a saprophyte and there is no complete sequence available for a plant pathogenic fungus in public databases. However, freely accessible entire genome sequences for both plant pathogenic fungi and their hosts are on the horizon. Sequence availability promises to revolutionize the rate at which data relevant to disease processes will be accrued. In this review we describe approaches currently applied to the study of plant pathogenic fungi and explore developments of potential future benefit with existing technologies not yet applied to this group of important organisms.

Fdb1 and Fdb2, Fusarium verticillioides loci necessary for detoxification of preformed antimicrobials from corn.

Fusarium verticillioides is a fungus of significant economic importance because of its deleterious effects on plant and animal health and on the quality of their products. Corn (Zea mays) is the primary host for F. verticillioides, and we have investigated the impact of the plant's antimicrobial compounds (DIMBOA, DIBOA, MBOA, and BOA) on fungal virulence and systemic colonization. F. verticillioides is able to metabolize these antimicrobials, and genetic analyses indicated two loci, Fdb1 and Fdb2, were involved in detoxification. Mutation at either locus caused sensitivity and no detoxification. In vitro physiological complementation assays resulted in detoxification of BOA and suggested that an unknown intermediate compound was produced. Production of the intermediate compound involved Fdbl, and a lesion in fdb2 preventing complete metabolism of BOA resulted in transformation of the intermediate into an unidentified metabolite. Based on genetic and physiological data, a branched detoxification pathway is proposed. Use of genetically characterized detoxifying and nondetoxifying strains indicated that detoxification of the corn antimicrobials was not a major virulence factor, since detoxification was not necessary for development of severe seedling blight or for infection and endophytic colonization of seedlings. Production of the antimicrobials does not appear to be a highly effective resistance mechanism against F. verticillioides.

Identification and complementation of a mutation to constitutive filamentous growth in Ustilago maydis.

Pathogenicity of the corn smut fungus Ustilago maydis involves the formation of a filamentous, infectious dikaryon by fusion of compatible, yeastlike haploid cells. The mating-type loci, a and b, regulate cell fusion and establishment of the dikaryotic cell type, respectively. On solid medium, compatibility at the mating-type loci, in particular heterozygosity at the b locus, is manifested by the formation of aerial hyphae on colonies formed by mating cells. We have employed this "fuzzy" phenotype to identify haploid mutants that constitutively form hyphal filaments and forego cell division by budding. A total of 125 such mutants have been isolated; characterization of one mutant (termed rem1-1) revealed that it can participate in infection of the host plant, although it must be paired with a compatible, wild-type mating partner. That is, mutation to the mycelial phenotype is not sufficient to allow a haploid strain to be pathogenic by itself. A cosmid has been isolated that restores the ability of an rem1-1 mutant to grow with a budding phenotype. Localization of the complementing region on cosmid DNA allowed the construction of an additional mutation by gene disruption. Coinoculation of plants with two compatible strains, each carrying the disruption mutation, gave greatly reduced disease symptoms. The analysis of the rem1 gene should contribute to an understanding of dimorphic growth and pathogenesis in U. maydis.

The Ustilago maydis ubc4 and ubc5 genes encode members of a MAP kinase cascade required for filamentous growth.

Ustilago maydis, the causal agent of corn smut disease, displays dimorphic growth in which it alternates between a budding haploid saprophyte and a filamentous dikaryotic pathogen. We are interested in identifying the genetic determinants of filamentous growth and pathogenicity in U. maydis. To do this we have taken a forward genetic approach. Earlier, we showed that haploid adenylate cyclase (uac1) mutants display a constitutively filamentous phenotype. Mutagenesis of a uac1 disruption strain allowed the isolation of a large number of budding suppressor mutants. These mutants are named ubc, for Ustilago bypass of cyclase, as they no longer require the production of cyclic AMP (cAMP) to grow in the budding morphology. Complementation of a subset of these suppressor mutants led to the identification of the ubc4 and ubc5 genes, which are required for filamentous growth and encode a MAP (mitogen-activated protein) kinase kinase kinase and a MAP kinase kinase, respectively. Evidence suggests that they are important in the pheromone response pathway and in pathogenicity. These results further support an important interplay of the cAMP and MAP kinase signal transduction pathways in the control of morphogenesis and pathogenicity in U. maydis.

Construction of a Tn7-lux system for gene expression studies in gram-negative bacteria.

A Tn7-lux system was developed for gene expression studies in Gram- bacteria. The plasmids constructed, pHSK728 and pHSK729, have the following features: (1) a promoterless Vibrio fischeri lux operon as a reporter system; (2) multiple cloning sites (MCS) ahead of the lux operon, in opposite orientation for the cloning of promoter fragments; (3) a transcriptional terminator ahead of the MCS and translational stop codons in all reading frames before the translational start of the luxC gene; (4) a streptomycin/spectinomycin-resistance encoding gene as a selection marker; and (5) Tn7 border sequences flanking the above elements, permitting the transposition of lux fusion constructs into bacterial genomes. The system was tested using the Escherichia coli lac promoter as well as the differentially regulated promoters of the avrD gene from Pseudomonas syringae pv. tomato and the pelE gene of Erwinia chrysanthemi EC16. Southern blot analysis showed that all fusion constructs had integrated into the host genomes in a single-copy, site-specific manner. The promoters of the avrD and pelE genes resulted in little or no light production when bacteria were grown in rich culture media, but high levels of induction were observed when the bacteria were grown in plant tissues. These results demonstrated that the Tn7-lux system provided a simple, sensitive assay of promoter activity in Gram- bacteria.

Three selectable markers for transformation of Ustilago maydis.

Although Ustilago maydis is readily amenable to molecular genetic experimentation, few antibiotic-resistance markers are available for DNA-mediated transformation. This poses constraints on experiments involving targeted gene disruption and complementation. To address this problem, we constructed vectors using one of three additional genes as dominant selectable markers for transformation. Two genes, sat-1 (encoding streptothricin acetyltransferase) and Sh-ble (encoding a phleomycin-resistance polypeptide), are of bacterial origin and have been engineered for expression in Ustilago sp. The third gene encodes an allele of U. maydis beta-tubulin that confers resistance to the fungicide benomyl.

Phylogeography and Genotype-Symptom Associations in Early and Late Season Infections of Canola by Sclerotinia sclerotiorum.

ABSTRACT Both typical late season stem infections and atypical early season rosette infections of canola, a relatively new crop in the southeastern United States, were caused by Sclerotinia sclerotiorum. The 51 DNA fingerprints (from 71 isolates) did not match any fingerprints from previous studies of canola or other crops. Single locus haplotypes from nuclear DNA sequences included 18 in the intergenic spacer (IGS) of the rRNA repeat, four in 44.11, six in translation elongation factor 1alpha, three in calmodulin (CAL), and two in chitin synthase 1. Contingency permutation testing for associations of infection type with DNA fingerprint, single- or multilocus haplotype, or hierarchically nested clades based on single locus haplotypes found significant association of haplotype with mycelial compatibility group and DNA fingerprint for all loci except CAL. Significant association of IGS haplotypes with symptom type was detected in one pathogen population. Southeastern U.S. canola was infected by both recently evolved, geographically dispersed pathogen genotypes and older, indigenous genotypes (Carbone and Kohn, 2001. Mol. Ecol. 10:947-964). Indigenous haplotypes are infection-type generalists, and the most frequently isolated from rosette infections. In contrast, haplotypes from the most recently evolved, dispersed population were associated one-to-one with infection type, with only the most recently evolved haplotypes infecting rosettes.

Whole-Genome Shotgun Sequence of Bacillus mojavensis Strain RRC101, an Endophytic Bacterium Antagonistic to the Mycotoxigenic Endophytic Fungus Fusarium verticillioides.

Here, we report the whole-genome shotgun sequence of Bacillus mojavensis strain RRC101, isolated from a maize kernel. This strain is antagonistic to the mycotoxigenic plant pathogen Fusarium verticillioides and grows within maize tissue, suggesting potential as an endophytic biocontrol agent.

The ubc2 gene of Ustilago maydis encodes a putative novel adaptor protein required for filamentous growth, pheromone response and virulence.

The Basidiomycete fungus Ustilago maydis causes corn smut disease and alternates between a budding haploid saprophyte and a filamentous dikaryotic pathogen. Previous work demonstrated that haploid adenylate cyclase (uac1) mutants display a constitutively filamentous phenotype. Suppressor mutants of a uac1 disruption strain, named ubc for Ustilago bypass of cyclase, no longer require cAMP for the budding morphology. The ubc2 gene was isolated by complementation and is required for filamentous growth. The deduced amino acid sequence encoded by ubc2 shows localized homology to Sterile Alpha Motif (SAM), Ras Association (RA) and Src homology 3 (SH3) protein-protein interaction domains. A K78E missense mutation within the SAM domain, revealed a genetic interaction between ubc2 and ubc4, a pheromone-responsive MAP kinase kinase kinase. This indicates involvement of ubc2 in the pheromone-responsive MAP kinase cascade and ubc2 is required for pheromone-responsive morphogenesis. The ubc2 gene is a critical virulence factor. Thus, ubc2 encodes a putative novel adaptor protein that may act directly upstream of the pheromone-responsive MAP kinase cascade in U. maydis.

Disruption of two genes for chitin synthase in the phytopathogenic fungus Ustilago maydis.

The phytopathogenic fungus Ustilago maydis exhibits a dimorphic transition in which non-pathogenic, yeast-like cells mate to form a pathogenic, filamentous dikaryon. Northern analysis indicated that two chitin synthase genes, chs1 and chs2, from U. maydis are expressed at similar levels in yeast-like cells and in cells undergoing the mating reaction leading to the filamentous cell type. A mutation was constructed in each of the chitin synthase genes by targeted gene disruption. Each mutant showed a reduction in the level of trypsin-activated enzyme activity, compared with a wild-type strain, but retained the wild-type morphology, the ability to mate and the ability to form the filamentous pathogenic cell type.

A MAP kinase encoded by the ubc3 gene of Ustilago maydis is required for filamentous growth and full virulence.

Ustilago maydis, the causal agent of corn smut disease, displays dimorphic growth in which it alternates between a budding haploid saprophyte and a filamentous dikaryotic pathogen. We are interested in identifying the genetic determinants of filamentous growth and pathogenicity in U. maydis. To do this, we have taken a forward genetic approach. Previously, we showed that haploid adenylate cyclase (uac1) mutants display a constitutively filamentous phenotype. Mutagenesis of a uac1 disruption strain allowed the isolation of a large number of budding suppressor mutants. These mutants are named ubc, for Ustilago bypass of cyclase, as they no longer require the production of cAMP to grow in the budding morphology. Complementation of one of these suppressor mutants led to the identification of ubc3, which is required for filamentous growth and encodes a MAP kinase most similar to those of the yeast pheromone response pathway. In addition to filamentous growth, the ubc3 gene is required for pheromone response and for full virulence. Mutations in the earlier identified fuz7 MAP kinase kinase also suppress the filamentous phenotype of the uac1 disruption mutant, adding evidence that both ubc3 and fuz7 are members of this same MAP kinase cascade. These results support an important interplay of the cAMP and MAP kinase signal transduction pathways in the control of morphogenesis and pathogenicity in U. maydis.

Characterization of two beta-tubulin genes from Geotrichum candidum.

The beta-tubulin genes G beta 1 and G beta 2 from the phytopathogenic hemiascomycete Geotrichum candidum were found to be highly diverged in amino acid sequence from those of other filamentous fungi. G beta 1 and G beta 2 were also divergent from each other, with the coding regions sharing only 66% nucleotide sequence homology and 64% amino acid identity. However, the proteins shared 82% similarity and only 25 of the 161 non-identical amino acid substitutions were non-conservative. The organization of G beta 1 is similar to other fungal beta-tubulin genes, but G beta 2 has several unusual features; it has 2 amino acid additions in the N-terminal 40 residues and must employ an uncommon 5' splice junction sequence in preference to an overlapping perfect consensus. The amino acid change found to confer benomyl resistance in Neurospora crassa was also present in G beta 2. G beta 1 has four introns which are located similarly to those of beta-tubulin genes in other fungi. G beta 2, however, has a single intron in a unique location. Translational fusions employing the 5' non-coding regions of the two Geotrichum beta-tubulin genes were made with the hygromycin phosphotransferase gene and shown to function in Schizosaccharomyces pombe and Trichoderma hamatum. However, G. candidum could not be transformed with these or other tested plasmids commonly used for fungal transformation.

The Ustilago maydis regulatory subunit of a cAMP-dependent protein kinase is required for gall formation in maize.

In the plant, filamentous growth is required for pathogenicity of the corn smut pathogen Ustilago maydis. Earlier, we identified a role for the cAMP signal transduction pathway in the switch between budding and filamentous growth for this fungus. A gene designated ubc1 (for Ustilago bypass of cyclase) was found to be required for filamentous growth and to encode the regulatory subunit of a cAMP-dependent protein kinase (PKA). Here, we show that ubc1 is important for the virulence of the pathogen. Specifically, ubc1 mutants are able to colonize maize plants and, like the wild-type pathogen, cause localized symptoms in association with the presence of hyphae. However, in contrast to plants infected with wild-type cells that often developed galls from initially chlorotic tissue, plants infected with the ubc1 mutant did not produce galls. These data suggest that PKA regulation is critical for the transition from saprophytic to pathogenic growth and from vegetative to reproductive development. Plate mating assays in which exogenous cAMP was applied suggested that the cAMP and b mating-type morphogenetic pathways may be coordinated.

MADS-box gene evolution beyond flowers: expression in pollen, endosperm, guard cells, roots and trichomes.

MADS-box genes encode transcriptional regulators involved in diverse aspects of plant development. Here we describe the cloning and mRNA spatio-temporal expression patterns of five new MADS-box genes from Arabidopsis: AGL16, AGL18, AGL19, AGL27 and AGL31. These genes will probably become important molecular tools for both evolutionary and functional analyses of vegetative structures. We mapped our data and previous expression patterns onto a new MADS-box phylogeny. These analyses suggest that the evolution of the MADS-box family has involved a rapid and simultaneous functional diversification in vegetative as well as reproductive structures. The hypothetical ancestral genes had broader expression patterns than more derived ones, which have been co-opted for putative specialized functions as suggested by their expression patterns. AGL27 and AGL31, which are closely related to the recently described flowering-time gene FLC (previously AGL25), are expressed in most plant tissues. AGL19 is specifically expressed in the outer layers of the root meristem (lateral root cap and epidermis) and in the central cylinder cells of mature roots. AGL18, which is most similar in sequence to the embryo-expressed AGL15 gene, is expressed in the endosperm and in developing male and female gametophytes, suggesting a role for AGL18 that is distinct from previously characterized MADS-box genes. Finally, AGL16 RNA accumulates in leaf guard cells and trichomes. Our new phylogeny reveals seven new monophyletic clades of MADS-box sequences not specific to flowers, suggesting that complex regulatory networks involving several MADS-box genes, similar to those that control flower development, underlie development of vegetative structures.

An ancestral MADS-box gene duplication occurred before the divergence of plants and animals.

Changes in genes encoding transcriptional regulators can alter development and are important components of the molecular mechanisms of morphological evolution. MADS-box genes encode transcriptional regulators of diverse and important biological functions. In plants, MADS-box genes regulate flower, fruit, leaf, and root development. Recent sequencing efforts in Arabidopsis have allowed a nearly complete sampling of the MADS-box gene family from a single plant, something that was lacking in previous phylogenetic studies. To test the long-suspected parallel between the evolution of the MADS-box gene family and the evolution of plant form, a polarized gene phylogeny is necessary. Here we suggest that a gene duplication ancestral to the divergence of plants and animals gave rise to two main lineages of MADS-box genes: TypeI and TypeII. We locate the root of the eukaryotic MADS-box gene family between these two lineages. A novel monophyletic group of plant MADS domains (AGL34 like) seems to be more closely related to previously identified animal SRF-like MADS domains to form TypeI lineage. Most other plant sequences form a clear monophyletic group with animal MEF2-like domains to form TypeII lineage. Only plant TypeII members have a K domain that is downstream of the MADS domain in most plant members previously identified. This suggests that the K domain evolved after the duplication that gave rise to the two lineages. Finally, a group of intermediate plant sequences could be the result of recombination events. These analyses may guide the search for MADS-box sequences in basal eukaryotes and the phylogenetic placement of new genes from other plant species.