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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 monolignol gene silencing induces super-susceptibility to wheat. Average penetration efficiency of Bgt in a wheat susceptible line due to gene silencing. After 4 h of bombardment, leaves were inoculated with a high density of Bgt conidiophores and successful entry into epidermal cells was 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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fig4: RNAi-mediated monolignol gene silencing induces super-susceptibility to wheat. Average penetration efficiency of Bgt in a wheat susceptible line due to gene silencing. After 4 h of bombardment, leaves were inoculated with a high density of Bgt conidiophores and successful entry into epidermal cells was 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: To characterize the functional role of monolignol genes, RNAi-mediated gene-silencing assays in susceptible wheat epidermal cells were conducted by biolistic transformation and subsequent microscopic analysis of the interaction of Bgt with transformed cells. Independent and combinatorial gene silencing with RNAi constructs of TmPAL, TmCAOMT, TmCCoAMT, and TmCAD were undertaken, with co-bombardment of GUS- or GFP–expressed constructs as positive controls for transformations. RNAi-based suppression enhanced the penetration efficiency of Bgt compared with the controls; the average penetration efficiency was increasing from 40% to about 60% by single gene silencing (Fig. 4). The highest penetration efficiency (64%) was obtained in the case of TmCAD silencing. Co-silencing of TmPAL+TmCAOMT, TmPAL+TmCAD, and TmCAOMT+TmCAD was performed in epidermal cells and, in all cases, penetration efficiency was enhanced over that of independent silencing. The marginally highest rate of penetration (78%) was observed with TmPAL+TmCAD co-silenced cells (Fig. 4).


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 monolignol gene silencing induces super-susceptibility to wheat. Average penetration efficiency of Bgt in a wheat susceptible line due to gene silencing. After 4 h of bombardment, leaves were inoculated with a high density of Bgt conidiophores and successful entry into epidermal cells was 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

fig4: RNAi-mediated monolignol gene silencing induces super-susceptibility to wheat. Average penetration efficiency of Bgt in a wheat susceptible line due to gene silencing. After 4 h of bombardment, leaves were inoculated with a high density of Bgt conidiophores and successful entry into epidermal cells was 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: To characterize the functional role of monolignol genes, RNAi-mediated gene-silencing assays in susceptible wheat epidermal cells were conducted by biolistic transformation and subsequent microscopic analysis of the interaction of Bgt with transformed cells. Independent and combinatorial gene silencing with RNAi constructs of TmPAL, TmCAOMT, TmCCoAMT, and TmCAD were undertaken, with co-bombardment of GUS- or GFP–expressed constructs as positive controls for transformations. RNAi-based suppression enhanced the penetration efficiency of Bgt compared with the controls; the average penetration efficiency was increasing from 40% to about 60% by single gene silencing (Fig. 4). The highest penetration efficiency (64%) was obtained in the case of TmCAD silencing. Co-silencing of TmPAL+TmCAOMT, TmPAL+TmCAD, and TmCAOMT+TmCAD was performed in epidermal cells and, in all cases, penetration efficiency was enhanced over that of independent silencing. The marginally highest rate of penetration (78%) was observed with TmPAL+TmCAD co-silenced cells (Fig. 4).

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