Plant Cell Rep (2004) 23:504–511 DOI 10.1007/s00299-004-0866-z BIOTIC AND ABIOTIC STRESS K. Fujita · Y. Matsuda · M. Wada · Y. Hirai · K. Mori · N. Moriura · T. Nonomura · K. Kakutani · H. Toyoda Powdery mildew pathogens can suppress the chitinase gene expression induced in detached inner epidermis of barley coleoptile Received: 3 May 2004 / Revised: 29 July 2004 / Accepted: 31 July 2004 / Published online: 22 September 2004  Springer-Verlag 2004 Abstract Two-step PCR (RT-PCR and nested PCR) was used to detect gene expression in powdery mildew pathogen-infected cells of detached inner epidermis of barley coleoptiles. Cellular contents of infected cells were microscopically suctioned with a micropipette and sub- jected to PCR. Triosephosphate isomerase and glyceral- dehyde-3-phosphate dehydrogenase genes involved in the glycolytic pathway and a stimulus-induced endochitinase gene were targeted, and their expression was determined by detecting cDNAs derived from spliced transcripts. The two gycolysis-related genes were constantly expressed in the tissue irrespective of pathogen inoculation. In con- trast, chitinase gene expression was induced in non-in- fected inner epidermis after detachment. After inocula- tion, this expression was selectively suppressed in patho- gen-invaded cells, in spite of continuous expression in non-invaded cells of the same epidermis. Thus, the pres- ent method enabled us to directly analyze transcripts in individual cells at the infection site and assess the capa- bility of the pathogen to regulate host gene expression. Keywords Micro-suctioning · Barley coleoptile epidermal cells · Two-step PCRs for single cells · Gene expression detection · Powdery mildew of barley Introduction Chitinous cell walls of filamentous fungal pathogens are a target of enzymatic digestion to suppress infection (Boller 1987), and the roles of endogenous and exogenous chitinases in defense have been discussed in terms of a promising strategy of disease control for major crop plants (Chet et al. 1993). For example, in powdery mil- dew fungi of barley, treatment with exogenous chitinases was effective in degrading the infection structures of the pathogen (Toyoda et al. 1991; Ikeda et al. 1996), and endochitinases were also produced in the leaves, inducing resistance to the powdery mildew (Kogel et al. 1994). In spite of the capacity of plants to produce protective en- dochitinases, compatible pathogens are not impeded in completion of their biotrophic life cycle on the host. This complicated situation has prompted speculation that fun- gal mechanisms exist that suppress plant antifungal func- tions such as chitinase production, while maintaining or accelerating basic functions for energy-productive me- tabolism in the host cell. In the present study, we found elicitation of chitinase gene expression in the inner epi- dermis detached from the outer epidermis of barley coleoptile tissue. This is an ideal molecular event with which to demonstrate active fungal function on host cells; selective suppression of chitinase gene expression in pathogen-invaded cells suggests positive action by the pathogen. In the present study, an experiment was de- signed to prove this possibility using a newly devised method: two-step-PCR (RT-PCR followed by nested PCR) of mature mRNAs in protoplasm micropipetted from single cells invaded by the powdery mildew patho- gen. Barley coleoptile epidermis powdery mildew fungus is an attractive model system with which to analyze cyto- logical and molecular events in host-parasite interactions. Since the coleoptile epidermis consists of pigment-free single cell layers, the cytological response of host cells attacked by the pathogen can be tracked under a micro- scope (Bushnell et al. 1967). In addition, molecular tech- niques have been successfully combined with such mi- Communicated by L. Peæa K. Fujita · Y. Matsuda ( ) ) · M. Wada · Y. Hirai · K. Mori · N. Moriura · T. Nonomura · H. Toyoda Laboratory of Plant Pathology and Biotechnology, Faculty of Agriculture, Kinki University, 3327-204 Nakamachi, Nara, 631-8505, Japan e-mail: ymatsuda@nara.kindai.ac.jp Tel.: +81-742-435223 Fax: +81-742-431155 K. Kakutani Pharmaceutical Research and Technology Institute, Kinki University, Higashi, Osaka, Japan