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

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.

Fig. 1.

The monolignol biosynthesis pathway. The grey route is the most favoured in angiosperms. All the enzymatic reactions presented in the pathway have been demonstrated at least in vitro. Because of the variety of isoenzymes and kinetic properties, alternative routes through the metabolic pathway may exist. For reactions with a question mark, the respective enzyme is unknown. PAL, phenylalanine ammonia lyase; C3H, p-coumarate 3-hydroxylase; CAOMT, caffeic acid O-methyltransferase; F5H, ferulic acid 5-hydroxylase; 4CL, 4-hydroxycinnamoyl-CoA ligase; CCoAMT, caffeoyl-CoA O-methyltransferase; CCR, cinnamoyl-CoA reductase; CAD, cinnamyl alcohol dehydrogenase; HCT, p-hydroxycinnamoyl-CoA reductase; C4H, cinnamate 4-hydoxylase.

Fig. 2.

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.

Fig. 3.

RT-PCR analysis of tissue-specific expression of monolignol biosynthesis in response to Blumeria graminis f.sp. tritici infection. TmSAMS1 (Bhuiyan et al., 2007) and TmPRX1 (Liu et al., 2005) genes were tested for comparison with the expression pattern of monolignol genes. Total RNA was isolated from epidermal (E) and mesophyll (M) tissues at 0 hpi and 24 hpi and reverse transcribed to cDNA. The expression of a chlorophyll a/b binding protein (TmCab) and a glyceraldehydes 3-phosphate dehydrogenase gene (TmGAPD) was used as an indicator of mesophyll contamination in epidermal tissues and a control of mRNA normalization, respectively.

Fig. 4.

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).

Fig. 5.

Fluorescence emission spectra of CWA and halo areas of monolignol gene-silenced cells. (A) Emission spectra of CWAs, (B) emission spectra of halo areas. A binary vector harbouring GUS was co-bombarded with RNAi vectors. HA, halo area; CWA: cell wall apposition. Fluorescence emission spectra and autofluorescence were measured by confocal laser scanning microscopy. Spectra shown are means of replicate measurements of at least ten papillae per treatment. Data shown represent the mean ±standard deviation of ten papillae per treatment. Scale bar corresponds to 10 μm. (This figure is available in colour at JXB online.)

Fig. 6.

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).

Fig. 7.

Effects of PAL and CAOMT inhibitors on the penetration ability of Bgt (A, B) and Bgh (C, D). (A, C) PAL inhibitor; (B, D) CAOMT inhibitor; (E) autofluorescence and (F) callose deposition in response to fungal interaction in CAOMT inhibitor-treated (right panel) and untreated (left panel) sample leaves. Con, conidia; AGT, appressorial germ tube. Scale bar corresponds to 10 μm. Wheat leaves were exposed to various concentrations of PAL and CAOMT inhibitors for 6 h prior to inoculation with Bgh. After inoculation, leaves were again placed on inhibitor solutions for 36 h. For observation of autofluorescence and callose deposition, leaves were collected at 20 hpi. Penetration efficiency was scored as described in the Materials and methods. Data shown represent mean ±standard deviation from three independent experiments in which 100 interaction sites were evaluated. Asterisks besides the columns indicate P <0.05 (Student's t test) compared with the negative control (0 mM inhibitor). (This figure is available in colour at JXB online.)