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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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Northern blot analysis of monolignol biosynthesis genes in response to Blumeria graminis f.sp. tritici infection. Total RNA was isolated at 0–144 hpi from 10-d-old primary leaves inoculated with B. graminis f.sp. tritici conidia. Uninoculated leaves were used as controls (CK). The transcript levels of the genes were analysed with 32P-labelled cDNA probes for TmPAL, TmCAOMT, TmF5H, TmCCoAMT, TmCCR, and TmCAD. Total RNA was loaded at 20 μg per lane and equal loading was monitored by methylene blue staining of ribosomal RNA.
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fig2: Northern blot analysis of monolignol biosynthesis genes in response to Blumeria graminis f.sp. tritici infection. Total RNA was isolated at 0–144 hpi from 10-d-old primary leaves inoculated with B. graminis f.sp. tritici conidia. Uninoculated leaves were used as controls (CK). The transcript levels of the genes were analysed with 32P-labelled cDNA probes for TmPAL, TmCAOMT, TmF5H, TmCCoAMT, TmCCR, and TmCAD. Total RNA was loaded at 20 μg per lane and equal loading was monitored by methylene blue staining of ribosomal RNA.

Mentions: The temporal expression pattern of the eight monolignol genes, in response to powdery mildew infection of a pair of susceptible and resistant wheat lines, was assessed over a 144 h post inoculation (hpi) period by Northern blotting (Fig. 2). The gel blot data show that the steady-state level of TmPAL, TmCCR, TmCCoAMT, TmF5H, TmCAOMT, and TmCAD transcripts were accumulated in response to infection but at different time points in the susceptible line. TmPAL and TmCCR showed the earliest expression at 3 hpi, that was also observed with a peroxidase gene, TmPRX1, in a previous study (Liu et al., 2005). Transcripts of TmCAOMT and TmCAD were strongly accumulated at 6 hpi, followed by a slight decrease at 12 hpi and then a sharp increase at 24 hpi. A similar expression pattern was also observed with TmSAMS1, a key gene of the methyl cycle in our previous study (Bhuiyan et al., 2007). The expression peaks at 6 hpi and 24 hpi coincide with attempted penetration time points of primary (4–6 hpi) and appressorial germ tubes (16–24 hpi), respectively (Collinge et al., 2002). Transcripts of TmCCoAMT were accumulated by 6 hpi, were sharply higher at 12 hpi, decreased through to 96 h before increasing again at 120 hpi and 144 hpi. Expression of TmF5H was induced by 24 hpi, and remained induced through to 120 hpi. Transcripts of Tm4CL (4-hydroxycinnamoyl-CoA ligase) and TmC3H (p-coumarate 3-hydroxylase) were not accumulated by Bgt infection in the susceptible line (data not shown).


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)

Northern blot analysis of monolignol biosynthesis genes in response to Blumeria graminis f.sp. tritici infection. Total RNA was isolated at 0–144 hpi from 10-d-old primary leaves inoculated with B. graminis f.sp. tritici conidia. Uninoculated leaves were used as controls (CK). The transcript levels of the genes were analysed with 32P-labelled cDNA probes for TmPAL, TmCAOMT, TmF5H, TmCCoAMT, TmCCR, and TmCAD. Total RNA was loaded at 20 μg per lane and equal loading was monitored by methylene blue staining of ribosomal RNA.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

fig2: Northern blot analysis of monolignol biosynthesis genes in response to Blumeria graminis f.sp. tritici infection. Total RNA was isolated at 0–144 hpi from 10-d-old primary leaves inoculated with B. graminis f.sp. tritici conidia. Uninoculated leaves were used as controls (CK). The transcript levels of the genes were analysed with 32P-labelled cDNA probes for TmPAL, TmCAOMT, TmF5H, TmCCoAMT, TmCCR, and TmCAD. Total RNA was loaded at 20 μg per lane and equal loading was monitored by methylene blue staining of ribosomal RNA.
Mentions: The temporal expression pattern of the eight monolignol genes, in response to powdery mildew infection of a pair of susceptible and resistant wheat lines, was assessed over a 144 h post inoculation (hpi) period by Northern blotting (Fig. 2). The gel blot data show that the steady-state level of TmPAL, TmCCR, TmCCoAMT, TmF5H, TmCAOMT, and TmCAD transcripts were accumulated in response to infection but at different time points in the susceptible line. TmPAL and TmCCR showed the earliest expression at 3 hpi, that was also observed with a peroxidase gene, TmPRX1, in a previous study (Liu et al., 2005). Transcripts of TmCAOMT and TmCAD were strongly accumulated at 6 hpi, followed by a slight decrease at 12 hpi and then a sharp increase at 24 hpi. A similar expression pattern was also observed with TmSAMS1, a key gene of the methyl cycle in our previous study (Bhuiyan et al., 2007). The expression peaks at 6 hpi and 24 hpi coincide with attempted penetration time points of primary (4–6 hpi) and appressorial germ tubes (16–24 hpi), respectively (Collinge et al., 2002). Transcripts of TmCCoAMT were accumulated by 6 hpi, were sharply higher at 12 hpi, decreased through to 96 h before increasing again at 120 hpi and 144 hpi. Expression of TmF5H was induced by 24 hpi, and remained induced through to 120 hpi. Transcripts of Tm4CL (4-hydroxycinnamoyl-CoA ligase) and TmC3H (p-coumarate 3-hydroxylase) were not accumulated by Bgt infection in the susceptible line (data not shown).

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