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RNA-seq Transcriptome Response of Flax ( Linum usitatissimum L.) to the Pathogenic Fungus Fusarium oxysporum f. sp. lini

View Article: PubMed Central - PubMed

ABSTRACT

Fusarium oxysporum f. sp. lini is a hemibiotrophic fungus that causes wilt in flax. Along with rust, fusarium wilt has become an important factor in flax production worldwide. Resistant flax cultivars have been used to manage the disease, but the resistance varies, depending on the interactions between specific cultivars and isolates of the pathogen. This interaction has a strong molecular basis, but no genomic information is available on how the plant responds to attempted infection, to inform breeding programs on potential candidate genes to evaluate or improve resistance across cultivars. In the current study, disease progression in two flax cultivars [Crop Development Center (CDC) Bethune and Lutea], showed earlier disease symptoms and higher susceptibility in the later cultivar. Chitinase gene expression was also divergent and demonstrated and earlier molecular response in Lutea. The most resistant cultivar (CDC Bethune) was used for a full RNA-seq transcriptome study through a time course at 2, 4, 8, and 18 days post-inoculation (DPI). While over 100 genes were significantly differentially expressed at both 4 and 8 DPI, the broadest deployment of plant defense responses was evident at 18 DPI with transcripts of more than 1,000 genes responding to the treatment. These genes evidenced a reception and transduction of pathogen signals, a large transcriptional reprogramming, induction of hormone signaling, activation of pathogenesis-related genes, and changes in secondary metabolism. Among these, several key genes that consistently appear in studies of plant-pathogen interactions, had increased transcript abundance in our study, and constitute suitable candidates for resistance breeding programs. These included: an induced RPMI-induced protein kinase; transcription factors WRKY3, WRKY70, WRKY75, MYB113, and MYB108; the ethylene response factors ERF1 and ERF14; two genes involved in auxin/glucosinolate precursor synthesis (CYP79B2 and CYP79B3); the flavonoid-related enzymes chalcone synthase, dihydroflavonol reductase and multiple anthocyanidin synthases; and a peroxidase implicated in lignin formation (PRX52). Additionally, regulation of some genes indicated potential pathogen manipulation to facilitate infection; these included four disease resistance proteins that were repressed, indole acetic acid amido/amino hydrolases which were upregulated, activated expansins and glucanases, amino acid transporters and aquaporins, and finally, repression of major latex proteins.

No MeSH data available.


Related in: MedlinePlus

Model depicting plant defense of flax upon Foln inoculation. Heatmaps for log2-fold gene expression changes at 2, 4, 8, and 18 DPI are shown besides each major gene group analyzed. The full deployment of plant defense is evidenced 18 DPI as seen in the forth column of the heatmaps. (1) During fungal attack, Fusarium oxysporum liberates elicitors (pathogen associated molecular patterns – PAMPs-), effectors and fungal proteases (which are also considered effectors) to facilitate infection. (2) Membrane receptors including receptor-like kinases (RLKs), an NBS-LRR (R-genes) interact with the PAMPs and effectors, respectively, causing downstream changes in phosphorylation of kinases (e.g., MAP kinases). (3) At the same time an influx of calcium causes changes in calcium-binding proteins that are also involved in signal transduction. (4) Regulation of transcription factors results in activation of hormone-related, defense, and secondary metabolism genes. (5) Presence of jasmonate (JA), ethylene (ET) biosynthetic genes indicates further signaling to other cells and feedback loops to activate more defense genes. (6) Protease inhibitors (PIs) neutralize fungal proteases while chitinases, thaumatins, and lipid transfer proteins (LTPs) act directly on the fungal cell wall or membrane. (7) Lignin precursors are created via phenylpropanoid metabolism and are polymerized into lignin by the action of laccases and peroxidases. (8) Peroxidases are also involved in the generation of reactive oxygen species (ROS) which are regulated by enzymes like glutathione S-transferases (GSTs). (9) Flavonoids and isoprenoids can act as antioxidants against ROS, or be directly translocated outside the cell by ATP-binding cassette (ABC) transporters to impair fungal function and growth. (10) Some unexpected regulation was found in some specific transcripts of several gene groups: auxin-related genes, major latex proteins (MLPs), cell wall modification proteins, major intrinsic proteins (MIPs), and amino acid permeases; the potential manipulation of the host by the pathogen to regulate such genes is indicated by a red arrow that parallels signal transduction and by the gene groups surrounded by dashed red lines (see text for explanation).
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Figure 5: Model depicting plant defense of flax upon Foln inoculation. Heatmaps for log2-fold gene expression changes at 2, 4, 8, and 18 DPI are shown besides each major gene group analyzed. The full deployment of plant defense is evidenced 18 DPI as seen in the forth column of the heatmaps. (1) During fungal attack, Fusarium oxysporum liberates elicitors (pathogen associated molecular patterns – PAMPs-), effectors and fungal proteases (which are also considered effectors) to facilitate infection. (2) Membrane receptors including receptor-like kinases (RLKs), an NBS-LRR (R-genes) interact with the PAMPs and effectors, respectively, causing downstream changes in phosphorylation of kinases (e.g., MAP kinases). (3) At the same time an influx of calcium causes changes in calcium-binding proteins that are also involved in signal transduction. (4) Regulation of transcription factors results in activation of hormone-related, defense, and secondary metabolism genes. (5) Presence of jasmonate (JA), ethylene (ET) biosynthetic genes indicates further signaling to other cells and feedback loops to activate more defense genes. (6) Protease inhibitors (PIs) neutralize fungal proteases while chitinases, thaumatins, and lipid transfer proteins (LTPs) act directly on the fungal cell wall or membrane. (7) Lignin precursors are created via phenylpropanoid metabolism and are polymerized into lignin by the action of laccases and peroxidases. (8) Peroxidases are also involved in the generation of reactive oxygen species (ROS) which are regulated by enzymes like glutathione S-transferases (GSTs). (9) Flavonoids and isoprenoids can act as antioxidants against ROS, or be directly translocated outside the cell by ATP-binding cassette (ABC) transporters to impair fungal function and growth. (10) Some unexpected regulation was found in some specific transcripts of several gene groups: auxin-related genes, major latex proteins (MLPs), cell wall modification proteins, major intrinsic proteins (MIPs), and amino acid permeases; the potential manipulation of the host by the pathogen to regulate such genes is indicated by a red arrow that parallels signal transduction and by the gene groups surrounded by dashed red lines (see text for explanation).

Mentions: Based on our observations of the flax transcriptome, we propose a model of the flax defense response to F. oxysporum f. sp. lini (Figure 5). Upon interaction, the fungus potentially liberates PAMPs (pathogen -associated molecular patterns), which are detected by membrane receptors like RLKs (PRRs), triggering innate immune responses via PTI (PAMP-triggered immunity). At the same time, effectors that act as virulence factors are probably deployed and detected by R-genes (or the R-genes detect changes in the interaction of the effectors with other cell components), in a gene-for-gene resistance fashion (vertical resistance), resulting in ETI (effecter-triggered immunity). A gene with similarity to RIPK from Arabidopsis was part of this response and may be an important component of resistance (Liu et al., 2011), while four disease resistance proteins downregulated throughout the time course represent interesting targets to study potential immune suppression by the pathogen, probably via small RNAs. The multitude of signal receptor genes indicate that flax (CDC Bethune) uses both non-specific and isolate-specific defenses to deter the pathogen, and therefore can activate both, a general immune response (e.g., PR genes), as well as a HR.


RNA-seq Transcriptome Response of Flax ( Linum usitatissimum L.) to the Pathogenic Fungus Fusarium oxysporum f. sp. lini
Model depicting plant defense of flax upon Foln inoculation. Heatmaps for log2-fold gene expression changes at 2, 4, 8, and 18 DPI are shown besides each major gene group analyzed. The full deployment of plant defense is evidenced 18 DPI as seen in the forth column of the heatmaps. (1) During fungal attack, Fusarium oxysporum liberates elicitors (pathogen associated molecular patterns – PAMPs-), effectors and fungal proteases (which are also considered effectors) to facilitate infection. (2) Membrane receptors including receptor-like kinases (RLKs), an NBS-LRR (R-genes) interact with the PAMPs and effectors, respectively, causing downstream changes in phosphorylation of kinases (e.g., MAP kinases). (3) At the same time an influx of calcium causes changes in calcium-binding proteins that are also involved in signal transduction. (4) Regulation of transcription factors results in activation of hormone-related, defense, and secondary metabolism genes. (5) Presence of jasmonate (JA), ethylene (ET) biosynthetic genes indicates further signaling to other cells and feedback loops to activate more defense genes. (6) Protease inhibitors (PIs) neutralize fungal proteases while chitinases, thaumatins, and lipid transfer proteins (LTPs) act directly on the fungal cell wall or membrane. (7) Lignin precursors are created via phenylpropanoid metabolism and are polymerized into lignin by the action of laccases and peroxidases. (8) Peroxidases are also involved in the generation of reactive oxygen species (ROS) which are regulated by enzymes like glutathione S-transferases (GSTs). (9) Flavonoids and isoprenoids can act as antioxidants against ROS, or be directly translocated outside the cell by ATP-binding cassette (ABC) transporters to impair fungal function and growth. (10) Some unexpected regulation was found in some specific transcripts of several gene groups: auxin-related genes, major latex proteins (MLPs), cell wall modification proteins, major intrinsic proteins (MIPs), and amino acid permeases; the potential manipulation of the host by the pathogen to regulate such genes is indicated by a red arrow that parallels signal transduction and by the gene groups surrounded by dashed red lines (see text for explanation).
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Figure 5: Model depicting plant defense of flax upon Foln inoculation. Heatmaps for log2-fold gene expression changes at 2, 4, 8, and 18 DPI are shown besides each major gene group analyzed. The full deployment of plant defense is evidenced 18 DPI as seen in the forth column of the heatmaps. (1) During fungal attack, Fusarium oxysporum liberates elicitors (pathogen associated molecular patterns – PAMPs-), effectors and fungal proteases (which are also considered effectors) to facilitate infection. (2) Membrane receptors including receptor-like kinases (RLKs), an NBS-LRR (R-genes) interact with the PAMPs and effectors, respectively, causing downstream changes in phosphorylation of kinases (e.g., MAP kinases). (3) At the same time an influx of calcium causes changes in calcium-binding proteins that are also involved in signal transduction. (4) Regulation of transcription factors results in activation of hormone-related, defense, and secondary metabolism genes. (5) Presence of jasmonate (JA), ethylene (ET) biosynthetic genes indicates further signaling to other cells and feedback loops to activate more defense genes. (6) Protease inhibitors (PIs) neutralize fungal proteases while chitinases, thaumatins, and lipid transfer proteins (LTPs) act directly on the fungal cell wall or membrane. (7) Lignin precursors are created via phenylpropanoid metabolism and are polymerized into lignin by the action of laccases and peroxidases. (8) Peroxidases are also involved in the generation of reactive oxygen species (ROS) which are regulated by enzymes like glutathione S-transferases (GSTs). (9) Flavonoids and isoprenoids can act as antioxidants against ROS, or be directly translocated outside the cell by ATP-binding cassette (ABC) transporters to impair fungal function and growth. (10) Some unexpected regulation was found in some specific transcripts of several gene groups: auxin-related genes, major latex proteins (MLPs), cell wall modification proteins, major intrinsic proteins (MIPs), and amino acid permeases; the potential manipulation of the host by the pathogen to regulate such genes is indicated by a red arrow that parallels signal transduction and by the gene groups surrounded by dashed red lines (see text for explanation).
Mentions: Based on our observations of the flax transcriptome, we propose a model of the flax defense response to F. oxysporum f. sp. lini (Figure 5). Upon interaction, the fungus potentially liberates PAMPs (pathogen -associated molecular patterns), which are detected by membrane receptors like RLKs (PRRs), triggering innate immune responses via PTI (PAMP-triggered immunity). At the same time, effectors that act as virulence factors are probably deployed and detected by R-genes (or the R-genes detect changes in the interaction of the effectors with other cell components), in a gene-for-gene resistance fashion (vertical resistance), resulting in ETI (effecter-triggered immunity). A gene with similarity to RIPK from Arabidopsis was part of this response and may be an important component of resistance (Liu et al., 2011), while four disease resistance proteins downregulated throughout the time course represent interesting targets to study potential immune suppression by the pathogen, probably via small RNAs. The multitude of signal receptor genes indicate that flax (CDC Bethune) uses both non-specific and isolate-specific defenses to deter the pathogen, and therefore can activate both, a general immune response (e.g., PR genes), as well as a HR.

View Article: PubMed Central - PubMed

ABSTRACT

Fusarium oxysporum f. sp. lini is a hemibiotrophic fungus that causes wilt in flax. Along with rust, fusarium wilt has become an important factor in flax production worldwide. Resistant flax cultivars have been used to manage the disease, but the resistance varies, depending on the interactions between specific cultivars and isolates of the pathogen. This interaction has a strong molecular basis, but no genomic information is available on how the plant responds to attempted infection, to inform breeding programs on potential candidate genes to evaluate or improve resistance across cultivars. In the current study, disease progression in two flax cultivars [Crop Development Center (CDC) Bethune and Lutea], showed earlier disease symptoms and higher susceptibility in the later cultivar. Chitinase gene expression was also divergent and demonstrated and earlier molecular response in Lutea. The most resistant cultivar (CDC Bethune) was used for a full RNA-seq transcriptome study through a time course at 2, 4, 8, and 18 days post-inoculation (DPI). While over 100 genes were significantly differentially expressed at both 4 and 8 DPI, the broadest deployment of plant defense responses was evident at 18 DPI with transcripts of more than 1,000 genes responding to the treatment. These genes evidenced a reception and transduction of pathogen signals, a large transcriptional reprogramming, induction of hormone signaling, activation of pathogenesis-related genes, and changes in secondary metabolism. Among these, several key genes that consistently appear in studies of plant-pathogen interactions, had increased transcript abundance in our study, and constitute suitable candidates for resistance breeding programs. These included: an induced RPMI-induced protein kinase; transcription factors WRKY3, WRKY70, WRKY75, MYB113, and MYB108; the ethylene response factors ERF1 and ERF14; two genes involved in auxin/glucosinolate precursor synthesis (CYP79B2 and CYP79B3); the flavonoid-related enzymes chalcone synthase, dihydroflavonol reductase and multiple anthocyanidin synthases; and a peroxidase implicated in lignin formation (PRX52). Additionally, regulation of some genes indicated potential pathogen manipulation to facilitate infection; these included four disease resistance proteins that were repressed, indole acetic acid amido/amino hydrolases which were upregulated, activated expansins and glucanases, amino acid transporters and aquaporins, and finally, repression of major latex proteins.

No MeSH data available.


Related in: MedlinePlus