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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.


Root sections of Lutea plants 22 DPI. The control plant (inoculated with water) on the top shows no signs of infection while the treated plant (isolate #81 inoculum) on the bottom has hyphae (indicated by arrowheads) colonizing the cortical cells (CC) and the xylem vessels (XV). Sections of 12 μm were stained with toluidine blue.
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Figure 3: Root sections of Lutea plants 22 DPI. The control plant (inoculated with water) on the top shows no signs of infection while the treated plant (isolate #81 inoculum) on the bottom has hyphae (indicated by arrowheads) colonizing the cortical cells (CC) and the xylem vessels (XV). Sections of 12 μm were stained with toluidine blue.

Mentions: CDC Bethune is an elite, brown-seeded linseed cultivar of flax that is widely grown in Canada and has been reported to have moderate resistance to fusarium wilt (Rowland et al., 2002). To confirm that CDC Bethune was relatively resistant, we conducted preliminary experiments with a panel of linseed varieties selected in consultation with a flax pathologist (Khalid Rashid, personal communication), and identified Lutea (a yellow-seeded variety) as a candidate cultivar that could differ in fusarium wilt resistance from CDC Bethune. We inoculated both cultivars with two F. oxysporum f. sp. lini isolates (#65 and #81) that demonstrated high spore viability/germination (not shown). CDC Bethune plants generally did not show any symptoms until 22 DPI, but wilting was evident in plants inoculated with isolate #81 (Figure 1). In Lutea plants, disease symptoms appeared earlier (8 DPI) than in CDC Bethune (22 DPI) and consequently the disease state was more advanced at 22 DPI, with some plants having undergone complete necrosis (Figure 1F). Disease symptoms recorded at 22 DPI included yellowing of leaves, brown spots on leaves, wilting, necrosis, and root browning. While most of these characteristics were variable and some infected plants presented little or no symptoms, root browning (represented as a general brown-ashy appearance indicative of rot) was a consistent symptom of disease in both cultivars and with both fungal isolates (Figure 2A). When using shoot length to assess the influence of the fungal inoculations on plant growth (Kroes et al., 1998b), there was a significant difference between the shoot lengths of control plants when compared with the lengths of isolate #81 inoculated Lutea plants (Figure 2B); nevertheless both cultivars had a 13% shoot length reduction with isolate #81 at 22 DPI. Together, these results showed that isolate #81 was the most aggressive F. oxysporum f. sp. lini isolate, and that CDC Bethune was more resistant to F. oxysporum f. sp. lini than Lutea, under our experimental conditions. We were able to re-isolate the fungus from surface-sterilized roots of previously inoculated plants of both CDC Bethune and Lutea (Supplementary Figures S1A,B). Colony and spore morphologies were consistent with the original inocula (Supplementary Figures S1C and S2). As further evidence of infection, we also stained sections of inoculated roots with toluidine blue. Hyphal development in root sections was advanced at 22 DPI, at which point hyphae had colonized the cortical cells and penetrated xylem vessels (Figure 3).


RNA-seq Transcriptome Response of Flax ( Linum usitatissimum L.) to the Pathogenic Fungus Fusarium oxysporum f. sp. lini
Root sections of Lutea plants 22 DPI. The control plant (inoculated with water) on the top shows no signs of infection while the treated plant (isolate #81 inoculum) on the bottom has hyphae (indicated by arrowheads) colonizing the cortical cells (CC) and the xylem vessels (XV). Sections of 12 μm were stained with toluidine blue.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
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Figure 3: Root sections of Lutea plants 22 DPI. The control plant (inoculated with water) on the top shows no signs of infection while the treated plant (isolate #81 inoculum) on the bottom has hyphae (indicated by arrowheads) colonizing the cortical cells (CC) and the xylem vessels (XV). Sections of 12 μm were stained with toluidine blue.
Mentions: CDC Bethune is an elite, brown-seeded linseed cultivar of flax that is widely grown in Canada and has been reported to have moderate resistance to fusarium wilt (Rowland et al., 2002). To confirm that CDC Bethune was relatively resistant, we conducted preliminary experiments with a panel of linseed varieties selected in consultation with a flax pathologist (Khalid Rashid, personal communication), and identified Lutea (a yellow-seeded variety) as a candidate cultivar that could differ in fusarium wilt resistance from CDC Bethune. We inoculated both cultivars with two F. oxysporum f. sp. lini isolates (#65 and #81) that demonstrated high spore viability/germination (not shown). CDC Bethune plants generally did not show any symptoms until 22 DPI, but wilting was evident in plants inoculated with isolate #81 (Figure 1). In Lutea plants, disease symptoms appeared earlier (8 DPI) than in CDC Bethune (22 DPI) and consequently the disease state was more advanced at 22 DPI, with some plants having undergone complete necrosis (Figure 1F). Disease symptoms recorded at 22 DPI included yellowing of leaves, brown spots on leaves, wilting, necrosis, and root browning. While most of these characteristics were variable and some infected plants presented little or no symptoms, root browning (represented as a general brown-ashy appearance indicative of rot) was a consistent symptom of disease in both cultivars and with both fungal isolates (Figure 2A). When using shoot length to assess the influence of the fungal inoculations on plant growth (Kroes et al., 1998b), there was a significant difference between the shoot lengths of control plants when compared with the lengths of isolate #81 inoculated Lutea plants (Figure 2B); nevertheless both cultivars had a 13% shoot length reduction with isolate #81 at 22 DPI. Together, these results showed that isolate #81 was the most aggressive F. oxysporum f. sp. lini isolate, and that CDC Bethune was more resistant to F. oxysporum f. sp. lini than Lutea, under our experimental conditions. We were able to re-isolate the fungus from surface-sterilized roots of previously inoculated plants of both CDC Bethune and Lutea (Supplementary Figures S1A,B). Colony and spore morphologies were consistent with the original inocula (Supplementary Figures S1C and S2). As further evidence of infection, we also stained sections of inoculated roots with toluidine blue. Hyphal development in root sections was advanced at 22 DPI, at which point hyphae had colonized the cortical cells and penetrated xylem vessels (Figure 3).

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.