6.161
INDUCTION OF SYSTEMIC ACQUIRED RESISTANCE IN WHEAT BY Pseudomonas aeruginosa
S MUYANGA 1, N KOEDAM 1, P CORNELIS 2, M HÖFTE 3 and M VAUTERIN 1.
1Inst. of Mol. Biology, University of Brussel, 65 Paardenstraat, B-1640 Sint Genesius Rode, Belgium; 2Vlaams Interuniversitair Instituut voor Biotech. (V.I.B.), Dept. Imm. Paras. and Ultr., 65 Paardenstraat, B-1640 Sint Genesius Rode, Belgium, and 3 Lab of Phytopathology, University of Gent, Coupure Links 653, B-9000 Gent, Belgium.

Background and objectives
Colonisation of wheat roots by Pseudomonas aeruginosa bacteria induces systemic acquired resistance (SAR). In vivo plant protection experiments involving Erysiphe graminis (powdery mildew) and Helminthosporium sativum (spot blotch) showed a significant reduction in folial fungal infection as a result of wheat root bacterisation. Apoplast protein analysis on SDS PAGE revealed two induced proteins and disappearance of one protein due to root bacterisation. Initial experiments gave rise to the isolation of a 0.5 kb cDNA from differentially expressed mRNAs. Northern blot analysis showed an hybridising band of about 1 kb. Southern blot analysis of cDNA pool, from both bacterised and nonbacterised plants, subjected to PCR amplification, showed strong hybridisation with cDNA from bacterised plants while a weak signal was observed in nonbacterised cDNA. This suggests that the 0.5 kb mRNA is enhanced as a result of bacterisation. However its involvement in SAR induction is to be confirmed. The aim of this work is to study the mechanism underlying SAR induction by root-colonising bacteria.


Materials and methods
Root colonisation of Spring wheat (Triticum aestivum) var. Nkwazi by Pseudomonas aeruginosa was investigated by using the Lac-Z reporter system. In vivo plant protection was studied by challenging both bacterised and unbacterised wheat plants with Erysiphe graminis (Powdery mildew) and Helminthosporium sativum (Spot blotch) infection on the foliage. Apoplast protein analysis was done to study the protein profiles in both bacterised and unbacterised plants. Genetic analysis involved RNA extraction and expression of differentially expressed mRNA and cDNA synthesis. One cDNA from induced mRNA was cloned and sequenced. Northern blot analysis was achieved. Southern hybridisation of the genomic DNA and PCR amplified cDNA pool from bacterised and unbacterised plant was performed with the differentially expressed cDNA isolated as a probe. Characterisation of the cDNA is currently underway.


Results and conclusions
It was shown that Pseudomonas aeruginosa is capable of efficiently colonising wheat roots. Bacterised wheat plants were significantly protected from Powdery mildew and Spot blotch by about 70%. SDS PAGE of apoplast protein indicated two induced proteins (24 and 25 kDa) and another 50 kDa protein disappeared. Northern hybridisation gave a strong signal only with RNA from bacterised wheat. A 0.5 kb differentially expressed cDNA was identified and it hybridised with genomic DNA and strongly with cDNA pool from bacterised plants while a weak signal was observed in unbacterised cDNA pool. Preliminary computer data base search of the 0.5 kb cDNA showed similarity to a transcription factor. From the data available so far it can be said that SAR is induced as a result of root bacterisation with Pseudomonas bacteria.


References
1. Tuzun S and Kloepper J, 1995. Induced Res. to Disease in Plants, 152-168. 2. Maurhofer M, Hase C, Meuwly P, Métraux JP, and Défago G, 1994. The American Phytopathological Society, Vol 84, No. 2, 139-146.