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RNA-seq analyses of gene expression in the microsclerotia of Verticillium dahliae.

Duressa D, Anchieta A, Chen D, Klimes A, Garcia-Pedrajas MD, Dobinson KF, Klosterman SJ - BMC Genomics (2013)

Bottom Line: Nearly 50% of the genes identified as differentially expressed in the MS library encode hypothetical proteins.Differential expression of selected genes observed by RNA-seq or microarray analysis was confirmed using RT-qPCR or Northern hybridizations.Collectively, the data acquired from these investigations provide additional insight into gene expression and molecular processes that occur during MS biogenesis and maturation in V. dahliae.

View Article: PubMed Central - HTML - PubMed

Affiliation: United States Department of Agriculture - Agricultural Research Service, Salinas, CA, USA. Steve.Klosterman@ars.usda.gov.

ABSTRACT

Background: The soilborne fungus, Verticillium dahliae, causes Verticillium wilt disease in plants. Verticillium wilt is difficult to control since V. dahliae is capable of persisting in the soil for 10 to 15 years as melanized microsclerotia, rendering crop rotation strategies for disease control ineffective. Microsclerotia of V. dahliae overwinter and germinate to produce infectious hyphae that give rise to primary infections. Consequently, microsclerotia formation, maintenance, and germination are critically important processes in the disease cycle of V. dahliae.

Results: To shed additional light on the molecular processes that contribute to microsclerotia biogenesis and melanin synthesis in V. dahliae, three replicate RNA-seq libraries were prepared from 10 day-old microsclerotia (MS)-producing cultures of V. dahliae, strain VdLs.17 (average = 52.23 million reads), and those not producing microsclerotia (NoMS, average = 50.58 million reads). Analyses of these libraries for differential gene expression revealed over 200 differentially expressed genes, including up-regulation of melanogenesis-associated genes tetrahydroxynaphthalene reductase (344-fold increase) and scytalone dehydratase (231-fold increase), and additional genes located in a 48.8 kilobase melanin biosynthetic gene cluster of strain VdLs.17. Nearly 50% of the genes identified as differentially expressed in the MS library encode hypothetical proteins. Additional comparative analyses of gene expression in V. dahliae, under growth conditions that promote or preclude microsclerotial development, were conducted using a microarray approach with RNA derived from V. dahliae strain Dvd-T5, and from the amicrosclerotial vdh1 strain. Differential expression of selected genes observed by RNA-seq or microarray analysis was confirmed using RT-qPCR or Northern hybridizations.

Conclusion: Collectively, the data acquired from these investigations provide additional insight into gene expression and molecular processes that occur during MS biogenesis and maturation in V. dahliae. The identified gene products could therefore potentially represent new targets for disease control through prevention of survival structure development.

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A 48.8 kb melanin biosynthetic cluster of genes in Verticillium dahliae. Genes up-regulated in the MS + culture are highlighted in yellow.
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Figure 2: A 48.8 kb melanin biosynthetic cluster of genes in Verticillium dahliae. Genes up-regulated in the MS + culture are highlighted in yellow.

Mentions: A number of known melanogenesis-related enzyme-encoding genes were identified by the data mining analysis as up-regulated in MS + culture vs. the NoMS culture, including THN reductase (VDAG_03665, 344-fold; VDAG_05181 87-fold), scytalone dehydratase (VDAG_03393, 231-fold), pigment biosynthesis protein Ayg1 (VDAG_04954, 165-fold), conidial yellow pigment biosynthesis PKS (VDAG_00190, 137-fold), two laccases (VDAG_00189, 111-fold, and VDAG_00034, 7-fold), versicolorin reductase (VDAG_00183, 41-fold), polyketide synthase (VDAG_00184, 23-fold), (Table 2, Additional file 4, #1-9). Among these, those encoding the PKS (VDAG_00190), versicolorin reductase (VDAG_00183), laccase (VDAG_00189) and a second polyketide synthase (VDAG_00184) are found clustered in a region spanning 48.8 kb on chromosome 2, supercontig 1 of the V. dahliae strain VdLs.17 genome (http://www.broadinstitute.org/annotation/genome/verticillium_dahliae) (Figure 2).


RNA-seq analyses of gene expression in the microsclerotia of Verticillium dahliae.

Duressa D, Anchieta A, Chen D, Klimes A, Garcia-Pedrajas MD, Dobinson KF, Klosterman SJ - BMC Genomics (2013)

A 48.8 kb melanin biosynthetic cluster of genes in Verticillium dahliae. Genes up-regulated in the MS + culture are highlighted in yellow.
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC3852263&req=5

Figure 2: A 48.8 kb melanin biosynthetic cluster of genes in Verticillium dahliae. Genes up-regulated in the MS + culture are highlighted in yellow.
Mentions: A number of known melanogenesis-related enzyme-encoding genes were identified by the data mining analysis as up-regulated in MS + culture vs. the NoMS culture, including THN reductase (VDAG_03665, 344-fold; VDAG_05181 87-fold), scytalone dehydratase (VDAG_03393, 231-fold), pigment biosynthesis protein Ayg1 (VDAG_04954, 165-fold), conidial yellow pigment biosynthesis PKS (VDAG_00190, 137-fold), two laccases (VDAG_00189, 111-fold, and VDAG_00034, 7-fold), versicolorin reductase (VDAG_00183, 41-fold), polyketide synthase (VDAG_00184, 23-fold), (Table 2, Additional file 4, #1-9). Among these, those encoding the PKS (VDAG_00190), versicolorin reductase (VDAG_00183), laccase (VDAG_00189) and a second polyketide synthase (VDAG_00184) are found clustered in a region spanning 48.8 kb on chromosome 2, supercontig 1 of the V. dahliae strain VdLs.17 genome (http://www.broadinstitute.org/annotation/genome/verticillium_dahliae) (Figure 2).

Bottom Line: Nearly 50% of the genes identified as differentially expressed in the MS library encode hypothetical proteins.Differential expression of selected genes observed by RNA-seq or microarray analysis was confirmed using RT-qPCR or Northern hybridizations.Collectively, the data acquired from these investigations provide additional insight into gene expression and molecular processes that occur during MS biogenesis and maturation in V. dahliae.

View Article: PubMed Central - HTML - PubMed

Affiliation: United States Department of Agriculture - Agricultural Research Service, Salinas, CA, USA. Steve.Klosterman@ars.usda.gov.

ABSTRACT

Background: The soilborne fungus, Verticillium dahliae, causes Verticillium wilt disease in plants. Verticillium wilt is difficult to control since V. dahliae is capable of persisting in the soil for 10 to 15 years as melanized microsclerotia, rendering crop rotation strategies for disease control ineffective. Microsclerotia of V. dahliae overwinter and germinate to produce infectious hyphae that give rise to primary infections. Consequently, microsclerotia formation, maintenance, and germination are critically important processes in the disease cycle of V. dahliae.

Results: To shed additional light on the molecular processes that contribute to microsclerotia biogenesis and melanin synthesis in V. dahliae, three replicate RNA-seq libraries were prepared from 10 day-old microsclerotia (MS)-producing cultures of V. dahliae, strain VdLs.17 (average = 52.23 million reads), and those not producing microsclerotia (NoMS, average = 50.58 million reads). Analyses of these libraries for differential gene expression revealed over 200 differentially expressed genes, including up-regulation of melanogenesis-associated genes tetrahydroxynaphthalene reductase (344-fold increase) and scytalone dehydratase (231-fold increase), and additional genes located in a 48.8 kilobase melanin biosynthetic gene cluster of strain VdLs.17. Nearly 50% of the genes identified as differentially expressed in the MS library encode hypothetical proteins. Additional comparative analyses of gene expression in V. dahliae, under growth conditions that promote or preclude microsclerotial development, were conducted using a microarray approach with RNA derived from V. dahliae strain Dvd-T5, and from the amicrosclerotial vdh1 strain. Differential expression of selected genes observed by RNA-seq or microarray analysis was confirmed using RT-qPCR or Northern hybridizations.

Conclusion: Collectively, the data acquired from these investigations provide additional insight into gene expression and molecular processes that occur during MS biogenesis and maturation in V. dahliae. The identified gene products could therefore potentially represent new targets for disease control through prevention of survival structure development.

Show MeSH
Related in: MedlinePlus