Publications by authors named "Roslyn D Noar"

8 Publications

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A polyketide synthase gene cluster associated with the sexual reproductive cycle of the banana pathogen, Pseudocercospora fijiensis.

PLoS One 2019 25;14(7):e0220319. Epub 2019 Jul 25.

Department of Plant and Microbial Biology, North Carolina State University, Raleigh, NC, United States of America.

Disease spread of Pseudocercospora fijiensis, causal agent of the black Sigatoka disease of banana, depends on ascospores produced through the sexual reproductive cycle. We used phylogenetic analysis to identify P. fijiensis homologs (PKS8-4 and Hybrid8-3) to the PKS4 polyketide synthases (PKS) from Neurospora crassa and Sordaria macrospora involved in sexual reproduction. These sequences also formed a clade with lovastatin, compactin, and betaenone-producing PKS sequences. Transcriptome analysis showed that both the P. fijiensis Hybrid8-3 and PKS8-4 genes have higher expression in infected leaf tissue compared to in culture. Domain analysis showed that PKS8-4 is more similar than Hybrid8-3 to PKS4. pPKS8-4:GFP transcriptional fusion transformants showed expression of GFP in flask-shaped structures in mycelial cultures as well as in crosses between compatible and incompatible mating types. Confocal microscopy confirmed expression in spermagonia in leaf substomatal cavities, consistent with a role in sexual reproduction. A disruption mutant of pks8-4 retained normal pathogenicity on banana, and no differences were observed in growth, conidial production, and spermagonia production. GC-MS profiling of the mutant and wild type did not identify differences in polyketide metabolites, but did identify changes in saturated fatty acid methyl esters and alkene and alkane derivatives. To our knowledge, this is the first report of a polyketide synthase pathway associated with spermagonia.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0220319PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6657885PMC
March 2020

A novel polyketide synthase gene cluster in the plant pathogenic fungus Pseudocercospora fijiensis.

PLoS One 2019 8;14(2):e0212229. Epub 2019 Feb 8.

Department of Plant and Microbial Biology, North Carolina State University, Raleigh, NC, United States of America.

Pseudocercospora fijiensis, causal agent of black Sigatoka of banana, produces polyketide synthase (PKS) pathways shown to be important in disease development by related Dothideomycete fungi. Genome analysis of the P. fijiensis PKS8-1 gene identified it as part of a gene cluster including genes encoding two transcription factors, a regulatory protein, a glyoxylase/beta-lactamase-like protein, an MFS transporter, a cytochrome P450, two aldo/keto reductases, a dehydrogenase, and a decarboxylase. Genome analysis of the related pathogens Pseudocercospora musae, Pseudocercospora eumusae, and Pseudocercospora pini-densiflorae, identified orthologous clusters containing a nearly identical combination of genes. Phylogenetic analysis of PKS8-1 identified homology to PKS proteins in the monodictyphenone and cladofulvin pathways in Aspergillus nidulans and Cladosporium fulvum, respectively. Analysis of clustered genes showed that the PKS8-1 cluster shares genes for enzymes involved in the production of the emodin intermediate in the monodictyphenone and cladofulvin pathways, but differs in many genes, suggesting production of a different metabolic product. Time course analysis of gene expression in infected banana showed up-regulation of PKS8-1 and four of eight clustered genes as early as 2 weeks post-inoculation and remaining high through 9 weeks. Overexpression of the pathway through constitutive expression of an aflR-like transcription factor gene in the cluster resulted in increased expression in culture of PKS8-1 as well as the four clustered genes that are up-regulated in infected plants. No differences were seen in timing or severity of disease symptoms with the overexpression strains relative to controls, however gene expression analysis showed no difference in expression in planta by an overexpression strain relative to controls. Thus constitutive expression of the aflR-like gene is not sufficient to upregulate the pathway above normal expression in planta. Pathway expression during all phases of disease development and conservation of the pathway in related Pseudocercospora species support a role for this pathway in disease.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0212229PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6368318PMC
November 2019

Transcriptome sequencing of Mycosphaerella fijiensis during association with Musa acuminata reveals candidate pathogenicity genes.

BMC Genomics 2016 08 30;17:690. Epub 2016 Aug 30.

Department of Plant and Microbial Biology, North Carolina State University, Raleigh, NC, 27695-7612, USA.

Background: Mycosphaerella fijiensis, causative agent of the black Sigatoka disease of banana, is considered the most economically damaging banana disease. Despite its importance, the genetics of pathogenicity are poorly understood. Previous studies have characterized polyketide pathways with possible roles in pathogenicity. To identify additional candidate pathogenicity genes, we compared the transcriptome of this fungus during the necrotrophic phase of infection with that during saprophytic growth in medium.

Results: Transcriptome analysis was conducted, and the functions of differentially expressed genes were predicted by identifying conserved domains, Gene Ontology (GO) annotation and GO enrichment analysis, Carbohydrate-Active EnZymes (CAZy) annotation, and identification of genes encoding effector-like proteins. The analysis showed that genes commonly involved in secondary metabolism have higher expression in infected leaf tissue, including genes encoding cytochrome P450s, short-chain dehydrogenases, and oxidoreductases in the 2-oxoglutarate and Fe(II)-dependent oxygenase superfamily. Other pathogenicity-related genes with higher expression in infected leaf tissue include genes encoding salicylate hydroxylase-like proteins, hydrophobic surface binding proteins, CFEM domain-containing proteins, and genes encoding secreted cysteine-rich proteins characteristic of effectors. More genes encoding amino acid transporters, oligopeptide transporters, peptidases, proteases, proteinases, sugar transporters, and proteins containing Domain of Unknown Function (DUF) 3328 had higher expression in infected leaf tissue, while more genes encoding inhibitors of peptidases and proteinases had higher expression in medium. Sixteen gene clusters with higher expression in leaf tissue were identified including clusters for the synthesis of a non-ribosomal peptide. A cluster encoding a novel fusicoccane was also identified. Two putative dispensable scaffolds were identified with a large proportion of genes with higher expression in infected leaf tissue, suggesting that they may play a role in pathogenicity. For two other scaffolds, no transcripts were detected in either condition, and PCR assays support the hypothesis that at least one of these scaffolds corresponds to a dispensable chromosome that is not required for survival or pathogenicity.

Conclusions: Our study revealed major changes in the transcriptome of Mycosphaerella fijiensis, when associating with its host compared to during saprophytic growth in medium. This analysis identified putative pathogenicity genes and also provides support for the existence of dispensable chromosomes in this fungus.
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http://dx.doi.org/10.1186/s12864-016-3031-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5006380PMC
August 2016

Bioinformatics Prediction of Polyketide Synthase Gene Clusters from Mycosphaerella fijiensis.

PLoS One 2016 7;11(7):e0158471. Epub 2016 Jul 7.

Department of Plant and Microbial Biology, North Carolina State University, Raleigh, North Carolina, 27695-7612, United States of America.

Mycosphaerella fijiensis, causal agent of black Sigatoka disease of banana, is a Dothideomycete fungus closely related to fungi that produce polyketides important for plant pathogenicity. We utilized the M. fijiensis genome sequence to predict PKS genes and their gene clusters and make bioinformatics predictions about the types of compounds produced by these clusters. Eight PKS gene clusters were identified in the M. fijiensis genome, placing M. fijiensis into the 23rd percentile for the number of PKS genes compared to other Dothideomycetes. Analysis of the PKS domains identified three of the PKS enzymes as non-reducing and two as highly reducing. Gene clusters contained types of genes frequently found in PKS clusters including genes encoding transporters, oxidoreductases, methyltransferases, and non-ribosomal peptide synthases. Phylogenetic analysis identified a putative PKS cluster encoding melanin biosynthesis. None of the other clusters were closely aligned with genes encoding known polyketides, however three of the PKS genes fell into clades with clusters encoding alternapyrone, fumonisin, and solanapyrone produced by Alternaria and Fusarium species. A search for homologs among available genomic sequences from 103 Dothideomycetes identified close homologs (>80% similarity) for six of the PKS sequences. One of the PKS sequences was not similar (< 60% similarity) to sequences in any of the 103 genomes, suggesting that it encodes a unique compound. Comparison of the M. fijiensis PKS sequences with those of two other banana pathogens, M. musicola and M. eumusae, showed that these two species have close homologs to five of the M. fijiensis PKS sequences, but three others were not found in either species. RT-PCR and RNA-Seq analysis showed that the melanin PKS cluster was down-regulated in infected banana as compared to growth in culture. Three other clusters, however were strongly upregulated during disease development in banana, suggesting that they may encode polyketides important in pathogenicity.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0158471PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4936691PMC
July 2017

Suppression of Arbuscule Degeneration in Medicago truncatula phosphate transporter4 Mutants is Dependent on the Ammonium Transporter 2 Family Protein AMT2;3.

Plant Cell 2015 Apr 3;27(4):1352-66. Epub 2015 Apr 3.

Boyce Thompson Institute for Plant Research, Tower Road, Ithaca, New York 14853

During arbuscular mycorrhizal (AM) symbiosis, the plant gains access to phosphate (Pi) and nitrogen delivered by its fungal symbiont. Transfer of mineral nutrients occurs at the interface between branched hyphae called arbuscules and root cortical cells. In Medicago truncatula, a Pi transporter, PT4, is required for symbiotic Pi transport, and in pt4, symbiotic Pi transport fails, arbuscules degenerate prematurely, and the symbiosis is not maintained. Premature arbuscule degeneration (PAD) is suppressed when pt4 mutants are nitrogen-deprived, possibly the result of compensation by PT8, a second AM-induced Pi transporter. However, PAD is also suppressed in nitrogen-starved pt4 pt8 double mutants, negating this hypothesis and furthermore indicating that in this condition, neither of these symbiotic Pi transporters is required for symbiosis. In M. truncatula, three AMT2 family ammonium transporters are induced during AM symbiosis. To test the hypothesis that suppression of PAD involves AMT2 transporters, we analyzed double and triple Pi and ammonium transporter mutants. ATM2;3 but not AMT2;4 was required for suppression of PAD in pt4, while AMT2;4, but not AMT2;3, complemented growth of a yeast ammonium transporter mutant. In summary, arbuscule life span is influenced by PT4 and ATM2;3, and their relative importance varies with the nitrogen status of the plant.
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http://dx.doi.org/10.1105/tpc.114.131144DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4558683PMC
April 2015

3D imaging and mechanical modeling of helical buckling in Medicago truncatula plant roots.

Proc Natl Acad Sci U S A 2012 Oct 24;109(42):16794-9. Epub 2012 Sep 24.

Department of Physics, Cornell University, Ithaca, NY 14853, USA.

We study the primary root growth of wild-type Medicago truncatula plants in heterogeneous environments using 3D time-lapse imaging. The growth medium is a transparent hydrogel consisting of a stiff lower layer and a compliant upper layer. We find that the roots deform into a helical shape just above the gel layer interface before penetrating into the lower layer. This geometry is interpreted as a combination of growth-induced mechanical buckling modulated by the growth medium and a simultaneous twisting near the root tip. We study the helical morphology as the modulus of the upper gel layer is varied and demonstrate that the size of the deformation varies with gel stiffness as expected by a mathematical model based on the theory of buckled rods. Moreover, we show that plant-to-plant variations can be accounted for by biomechanically plausible values of the model parameters.
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http://dx.doi.org/10.1073/pnas.1209287109DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3479456PMC
October 2012

Polar localization of a symbiosis-specific phosphate transporter is mediated by a transient reorientation of secretion.

Proc Natl Acad Sci U S A 2012 Mar 21;109(11):E665-72. Epub 2012 Feb 21.

Boyce Thompson Institute for Plant Research, Ithaca, NY 14853, USA.

The arbuscular mycorrhizal (AM) symbiosis, formed by land plants and AM fungi, evolved an estimated 400 million years ago and has been maintained in angiosperms, gymnosperms, pteridophytes, and some bryophytes as a strategy for enhancing phosphate acquisition. During AM symbiosis, the AM fungus colonizes the root cortical cells where it forms branched hyphae called arbuscules that function in nutrient exchange with the plant. Each arbuscule is enveloped in a plant membrane, the periarbuscular membrane, that contains a unique set of proteins including phosphate transporters such as Medicago truncatula MtPT4 [Javot et al., (2007) Proc Natl Acad Sci USA 104:1720-1725], which are essential for symbiotic phosphate transport. The periarbuscular membrane is physically continuous with the plasma membrane of the cortical cell, but MtPT4 and other periarbuscular membrane-resident proteins are located only in the domain around the arbuscule branches. Establishing the distinct protein composition of the periarbuscular membrane is critical for AM symbiosis, but currently the mechanism by which this composition is achieved is unknown. Here we investigate the targeting of MtPT4 to the periarbuscular membrane. By expressing MtPT4 and other plasma membrane proteins from promoters active at different phases of the symbiosis, we show that polar targeting of MtPT4 is mediated by precise temporal expression coupled with a transient reorientation of secretion and alterations in the protein cargo entering the secretory system of the colonized root cell. In addition, analysis of phosphate transporter mutants implicates the trans-Golgi network in phosphate transporter secretion.
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http://dx.doi.org/10.1073/pnas.1110215109DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3306687PMC
March 2012

Medicago truncatula mtpt4 mutants reveal a role for nitrogen in the regulation of arbuscule degeneration in arbuscular mycorrhizal symbiosis.

Plant J 2011 Dec 17;68(6):954-65. Epub 2011 Oct 17.

Boyce Thompson Institute for Plant Research, Ithaca, NY 14853, USA.

Plants acquire essential mineral nutrients such as phosphorus (P) and nitrogen (N) directly from the soil, but the majority of the vascular plants also gain access to these mineral nutrients through endosymbiotic associations with arbuscular mycorrhizal (AM) fungi. In AM symbiosis, the fungi deliver P and N to the root through branched hyphae called arbuscules. Previously we identified MtPT4, a Medicago truncatula phosphate transporter located in the periarbuscular membrane that is essential for symbiotic phosphate transport and for maintenance of the symbiosis. In mtpt4 mutants arbuscule degeneration occurs prematurely and symbiosis fails. Here, we show that premature arbuscule degeneration occurs in mtpt4 mutants even when the fungus has access to carbon from a nurse plant. Thus, carbon limitation is unlikely to be the primary cause of fungal death. Surprisingly, premature arbuscule degeneration is suppressed if mtpt4 mutants are deprived of nitrogen. In mtpt4 mutants with a low N status, arbuscule lifespan does not differ from that of the wild type, colonization of the mtpt4 root system occurs as in the wild type and the fungus completes its life cycle. Sulphur is another essential macronutrient delivered to the plant by the AM fungus; however, suppression of premature arbuscule degeneration does not occur in sulphur-deprived mtpt4 plants. The mtpt4 arbuscule phenotype is strongly correlated with shoot N levels. Analyses of an mtpt4-2 sunn-1 double mutant indicates that SUNN, required for N-mediated autoregulation of nodulation, is not involved. Together, the data reveal an unexpected role for N in the regulation of arbuscule lifespan in AM symbiosis.
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http://dx.doi.org/10.1111/j.1365-313X.2011.04746.xDOI Listing
December 2011