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Gene expression profiling and silencing reveal that monolignol biosynthesis plays a critical role in penetration defence in wheat against powdery mildew invasion.

Bhuiyan NH, Selvaraj G, Wei Y, King J - J. Exp. Bot. (2008)

Bottom Line: Co-silencing led to greater penetration of Bgt or Bgh than when the genes were silenced separately.Fluorescence emission spectra analyses revealed that gene silencing hampered host autofluorescence response at fungal contact sites.These results illustrate that monolignol biosynthesis is critically important for host defence against both appropriate and inappropriate pathogen invasion in wheat.

View Article: PubMed Central - PubMed

Affiliation: Department of Biology, University of Saskatchewan, Saskatoon, SK S7N 5E2, Canada. nazmul.bhuiyan@yahoo.com

ABSTRACT
Cell wall apposition (CWA) formation is one of the first lines of defence used by plants to halt invading fungi such as powdery mildew. Lignin is a complex polymer of hydroxylated and methoxylated phenylpropane units (monolignols) and lignification renders the cell wall more resistant to pathogen attack. The role of monolignol biosynthesis in CWA-mediated defence against powdery mildew penetration into cereals is demonstrated here using RNA interference (RNAi)-mediated gene silencing and enzyme-specific inhibitors. Thirteen cDNAs representing eight genes involved in monolignol biosynthesis were cloned from an expression sequence tag (EST) library derived from the epidermis of diploid wheat (Triticum monococcum) infected with Blumeria graminis f. sp. tritici (Bgt). Differential expression patterns were found for these genes in susceptible and resistant plants after infection. Transcripts of phenylalanine ammonia lyase (PAL), caffeic acid O-methyltransferase (CAOMT), ferulic acid hydroxylase (FAH), caffeoyl-CoA O-methyltransferase (CCoAMT), and cinnamyl alcohol dehydrogenase (CAD) were accumulated, particularly in the epidermis. RNAi-mediated transient gene silencing in the epidermis led to a higher penetration efficiency of Bgt than in the controls. Gene silencing also compromised penetration resistance to varying degrees with different genes against an inappropriate pathogen, B. graminis f. sp. hordei (Bgh). Co-silencing led to greater penetration of Bgt or Bgh than when the genes were silenced separately. Fluorescence emission spectra analyses revealed that gene silencing hampered host autofluorescence response at fungal contact sites. These results illustrate that monolignol biosynthesis is critically important for host defence against both appropriate and inappropriate pathogen invasion in wheat.

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RNAi-mediated gene silencing compromises penetration resistance of the non-host fungus, Blumeria graminis f.sp. hordei (Bgh). (A) Penetration efficiency of Bgh in the susceptible wheat line was evaluated by at least three independent experiments for each construct. Bars represent standard errors. (B) GUS expressing transformed cell that was inaccessible to Bgh (upper panel) and a TmCAOMT-silenced cell that was penetrated by Bgh (lower panel). The fungus formed a haustorium (H) and elongated secondary hyphae (ESH) on the leaf surface. Conidia were indicated by Con. After 4 h of bombardment, leaves were inoculated with a high density of Bgh conidiophores and successful entry into the epidermis cells were evaluated with microscopy as described in the Materials and methods. Data shown represent mean ±standard deviation from at least three experiments in which, as a minimum, 100 GUS-stained cells were evaluated. Asterisks besides columns indicate P <0.05 (Student's t test) compared to the negative control (GUS only).
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fig6: RNAi-mediated gene silencing compromises penetration resistance of the non-host fungus, Blumeria graminis f.sp. hordei (Bgh). (A) Penetration efficiency of Bgh in the susceptible wheat line was evaluated by at least three independent experiments for each construct. Bars represent standard errors. (B) GUS expressing transformed cell that was inaccessible to Bgh (upper panel) and a TmCAOMT-silenced cell that was penetrated by Bgh (lower panel). The fungus formed a haustorium (H) and elongated secondary hyphae (ESH) on the leaf surface. Conidia were indicated by Con. After 4 h of bombardment, leaves were inoculated with a high density of Bgh conidiophores and successful entry into the epidermis cells were evaluated with microscopy as described in the Materials and methods. Data shown represent mean ±standard deviation from at least three experiments in which, as a minimum, 100 GUS-stained cells were evaluated. Asterisks besides columns indicate P <0.05 (Student's t test) compared to the negative control (GUS only).

Mentions: Success in making wheat leaves significantly more susceptible to Bgt through the down-regulation of monolignol genes by the application of RNAi-mediated gene silencing, led to investigating whether this approach would lead to penetration by inappropriate or non-host pathogens. The independent gene silencing of TmPAL, TmCAOMT, TmCCoAMT, and TmCAD compromised penetration resistance to Bgh in wheat to varying degrees (Fig. 6A). No penetration was observed in the controls (Fig. 6A, B, upper panel). Figure 6B (lower panel) shows an example of the successful penetration of Bgh in a TmCAOMT-silenced cell of a wheat leaf where GUS was used as a marker gene. A GFP-expressing TmCAOMT-silenced cell was also susceptible to Bgh penetration (data not shown). The efficiency of Bgh penetration showed differences from those in the case of Bgt and individual gene silencing. The most efficient penetration was observed with TmCAOMT-silenced cells (11%). Co-silencing increased the penetration ability of Bgh with maximum penetration efficiency (15%) obtained when TmCAOMT and TmCAD were silenced together. Significantly higher efficiencies of penetration were also found with TmPAL+TmCAD (9%) and TmPAL+TmCAOMT (14%) silenced cells than when these genes were silenced separately (Fig. 6A).


Gene expression profiling and silencing reveal that monolignol biosynthesis plays a critical role in penetration defence in wheat against powdery mildew invasion.

Bhuiyan NH, Selvaraj G, Wei Y, King J - J. Exp. Bot. (2008)

RNAi-mediated gene silencing compromises penetration resistance of the non-host fungus, Blumeria graminis f.sp. hordei (Bgh). (A) Penetration efficiency of Bgh in the susceptible wheat line was evaluated by at least three independent experiments for each construct. Bars represent standard errors. (B) GUS expressing transformed cell that was inaccessible to Bgh (upper panel) and a TmCAOMT-silenced cell that was penetrated by Bgh (lower panel). The fungus formed a haustorium (H) and elongated secondary hyphae (ESH) on the leaf surface. Conidia were indicated by Con. After 4 h of bombardment, leaves were inoculated with a high density of Bgh conidiophores and successful entry into the epidermis cells were evaluated with microscopy as described in the Materials and methods. Data shown represent mean ±standard deviation from at least three experiments in which, as a minimum, 100 GUS-stained cells were evaluated. Asterisks besides columns indicate P <0.05 (Student's t test) compared to the negative control (GUS only).
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC2651457&req=5

fig6: RNAi-mediated gene silencing compromises penetration resistance of the non-host fungus, Blumeria graminis f.sp. hordei (Bgh). (A) Penetration efficiency of Bgh in the susceptible wheat line was evaluated by at least three independent experiments for each construct. Bars represent standard errors. (B) GUS expressing transformed cell that was inaccessible to Bgh (upper panel) and a TmCAOMT-silenced cell that was penetrated by Bgh (lower panel). The fungus formed a haustorium (H) and elongated secondary hyphae (ESH) on the leaf surface. Conidia were indicated by Con. After 4 h of bombardment, leaves were inoculated with a high density of Bgh conidiophores and successful entry into the epidermis cells were evaluated with microscopy as described in the Materials and methods. Data shown represent mean ±standard deviation from at least three experiments in which, as a minimum, 100 GUS-stained cells were evaluated. Asterisks besides columns indicate P <0.05 (Student's t test) compared to the negative control (GUS only).
Mentions: Success in making wheat leaves significantly more susceptible to Bgt through the down-regulation of monolignol genes by the application of RNAi-mediated gene silencing, led to investigating whether this approach would lead to penetration by inappropriate or non-host pathogens. The independent gene silencing of TmPAL, TmCAOMT, TmCCoAMT, and TmCAD compromised penetration resistance to Bgh in wheat to varying degrees (Fig. 6A). No penetration was observed in the controls (Fig. 6A, B, upper panel). Figure 6B (lower panel) shows an example of the successful penetration of Bgh in a TmCAOMT-silenced cell of a wheat leaf where GUS was used as a marker gene. A GFP-expressing TmCAOMT-silenced cell was also susceptible to Bgh penetration (data not shown). The efficiency of Bgh penetration showed differences from those in the case of Bgt and individual gene silencing. The most efficient penetration was observed with TmCAOMT-silenced cells (11%). Co-silencing increased the penetration ability of Bgh with maximum penetration efficiency (15%) obtained when TmCAOMT and TmCAD were silenced together. Significantly higher efficiencies of penetration were also found with TmPAL+TmCAD (9%) and TmPAL+TmCAOMT (14%) silenced cells than when these genes were silenced separately (Fig. 6A).

Bottom Line: Co-silencing led to greater penetration of Bgt or Bgh than when the genes were silenced separately.Fluorescence emission spectra analyses revealed that gene silencing hampered host autofluorescence response at fungal contact sites.These results illustrate that monolignol biosynthesis is critically important for host defence against both appropriate and inappropriate pathogen invasion in wheat.

View Article: PubMed Central - PubMed

Affiliation: Department of Biology, University of Saskatchewan, Saskatoon, SK S7N 5E2, Canada. nazmul.bhuiyan@yahoo.com

ABSTRACT
Cell wall apposition (CWA) formation is one of the first lines of defence used by plants to halt invading fungi such as powdery mildew. Lignin is a complex polymer of hydroxylated and methoxylated phenylpropane units (monolignols) and lignification renders the cell wall more resistant to pathogen attack. The role of monolignol biosynthesis in CWA-mediated defence against powdery mildew penetration into cereals is demonstrated here using RNA interference (RNAi)-mediated gene silencing and enzyme-specific inhibitors. Thirteen cDNAs representing eight genes involved in monolignol biosynthesis were cloned from an expression sequence tag (EST) library derived from the epidermis of diploid wheat (Triticum monococcum) infected with Blumeria graminis f. sp. tritici (Bgt). Differential expression patterns were found for these genes in susceptible and resistant plants after infection. Transcripts of phenylalanine ammonia lyase (PAL), caffeic acid O-methyltransferase (CAOMT), ferulic acid hydroxylase (FAH), caffeoyl-CoA O-methyltransferase (CCoAMT), and cinnamyl alcohol dehydrogenase (CAD) were accumulated, particularly in the epidermis. RNAi-mediated transient gene silencing in the epidermis led to a higher penetration efficiency of Bgt than in the controls. Gene silencing also compromised penetration resistance to varying degrees with different genes against an inappropriate pathogen, B. graminis f. sp. hordei (Bgh). Co-silencing led to greater penetration of Bgt or Bgh than when the genes were silenced separately. Fluorescence emission spectra analyses revealed that gene silencing hampered host autofluorescence response at fungal contact sites. These results illustrate that monolignol biosynthesis is critically important for host defence against both appropriate and inappropriate pathogen invasion in wheat.

Show MeSH
Related in: MedlinePlus