Expression of coordinately regulated defence response genes and analysis of their role in disease resistance in Medicago truncatula

Abstract

Microarray technology was used to identify the genes associated with disease defence responses in the model legume Medicago truncatula. Transcript profiles from M. truncatula cv. Jemalong genotype A17 leaves inoculated with Colletotrichum trifolii and Erysiphe pisi and roots infected with Phytophthora medicaginis were compared to identify the genes expressed in response to all three pathogens and genes unique to an interaction. The A17 genotype is resistant to C. trifolii and E. pisi, exhibiting a hypersensitive response after inoculation, and is moderately susceptible to P. medicaginis. Among the most strongly up-regulated genes in all three interactions were those encoding a hevein-like protein, thaumatin-like protein (TLP) and members of the pathogenesis response (PR)10 family. Transcripts of genes for enzymes in the phenylpropanoid pathway leading to the production of isoflavonoid phytoalexins increased dramatically in response to inoculation with the foliar pathogens. In P. medicaginis-inoculated roots, transcripts of genes in the phenylpropanoid pathway peaked at 5 days post-inoculation, when symptoms became visible. Transcript accumulation of three PR10 family members, a TLP and chalcone synthase (CHS) was assessed in M. truncatula genotype R108 plants. The R108 plants are resistant to C. trifolii and moderately susceptible to E. pisi and P. medicaginis. Transcript accumulation paralleled the stages of pathogen development. To evaluate the role of a TLP, a PR10 family member and CHS in disease resistance, transgenic R108 plants containing interfering RNA (RNAi) constructs were produced. Reduced expression of PR10 and TLP had no effect on the disease phenotype, whereas reduced expression of CHS resulted in increased susceptibility to necrotrophic pathogens.

Figure 1

Phylogenetic tree based on the alignment of the deduced amino acid sequences of pathogenesis response (PR)10 proteins from legumes and representative members of the monocot/pine, Solanaceae and tree allergen groups. Protein sequences were aligned with Clustal W. Based on the alignment, a phylogenetic tree was constructed by the neighbour‐joining method. The parsley PR1‐3 protein (X12573; PcPR1‐3) is included as the outgroup. Bootstrap values are given at branch points. The scale indicates genetic distance proportional to the amino acid substitutions per site. Sequence abbreviations of PR10 proteins are given with GenBank accession numbers in parentheses as follows: Arachis hypogaea PR10 (AAU81922, CAJ43118), Asparagus officinalis AoPR1 (X64452), Betula pendula Betv1 (CAA96547), Capsicum annuum PR10 (AF244121), Glycine max H4 (X60044), G. max PR10 (ABB73065), G. max SAM22 (X60043), Lupinus luteus LlPR10.1C (AAD55099), L. luteus PR10.2 (AAF77633), Malus×domestica Mald1.03B (AAX18314), Medicago sativa pprg2 (AJ320476), M. sativa PR10 (X98867), M. sativa SRG1 (U42752), M. truncatula PR10‐1 (Y08641), Oryza sativa PR10a (AF274850), Petroselinum cripsum PcRR1‐3 (X12573), Phaseolus vulgaris PvPR2 (P25986), Pinus pinaster PR‐10 (AJ291767), Pisum fulvum PR10 (U65425), P. sativum ABR17 (Z15128), P. sativum ABR18 (Z15127), P. sativum DRR49a (U31669), P. sativum DRRG49‐c (J03680), P. sativum PI49 (X13383), P. sativum PI176 (M18249), P. sativum RH2 (S74512), Solanum tuberosum pSTH‐2 (M25155), Zea mays PR‐10 (AY953127).

Figure 2

Phytophthora medicaginis DNA in roots of Medicago truncatula A17 and R108 as measured by quantitative real‐time polymerase chain reaction. Bars indicate standard deviation (n= 6).

Figure 3

Relative expression of pathogenesis response (PR)10, PR10.1, PR10.2, thaumatin‐like protein (TLP) and chalcone synthase (CHS) in Medicago truncatula R108 after pathogen inoculation by quantitative reverse transcriptase‐polymerase chain reaction (qRT‐PCR). Values are the average fold difference of inoculated plants relative to mock‐inoculated plants as determined by the ΔΔC _t method. Bars indicate standard deviation (n= 3). (a) Relative gene expression in leaves inoculated with Colletotrichum trifolii race 1. (b) Relative gene expression in leaves inoculated with Erysiphe pisi. (c) Relative gene expression in roots of plants inoculated with Phytophthora medicaginis.

Figure 4

Colletotrichum trifolii development on detached leaves of vector control Medicago truncatula and line CHS11 RNAi plants. Leaflets were placed in moist chambers, spot inoculated with a 6‐µL drop of spores at 7 × 10⁴ conidia/mL and held on the laboratory bench at 23 °C under ambient light conditions. At 48 h post‐inoculation, leaflets were fixed and stained with lactophenol trypan blue for light microscopy. Appressoria appear as brown melanized structures and mycelia stain blue. The vector control (R108 background) is resistant to C. trifolli.