75 Integrating Membrane Transport with Male Gametophyte ... - TAIR

277 MEKK1 Negatively Regulates Tissue Specific and Temperature Dependent Cell Death in
Arabidopsis
Kazuya Ichimura 1, 2 , Catarina Casais 1 , Scott Peck 1, 3 , Kazuo Shinozaki 2 , Ken Shirasu 1, 2
1
The Sainsbury Laboratory, John Innes Centre, Norwich, UK., 2 RIKEN Plant Science Center, Yokohama,
Japan., 3 Department of Biochemistry, University of Missouri Columbia, MO
Innate immunity signaling pathways in plants are regulated by mitogen-activated protein kinase (MAPK) cascades.
Using protoplast transient expression system, Arabidopsis MEKK1, a MAPK kinase kinase (MAPKKK) has been proposed
as a positive regulator of innate immunity signaling in the upstream of the MKK4, MKK5 (MAPKKs) - MPK3, MPK6
(MAPKs) pathway (1). In addition, yeast two-hybrid and complementation analyses and protoplast transient expression
experiment suggested that MEKK1 has a role in the upstream of the MKK1, MKK2 (MAPKKs) – MPK4 pathway (2,
3, 4). Flg22 treatment activates MPK3, MPK4, and MPK6 (1, 5). However, genetic evidence of MEKK1 involvement
in flg22-triggered MAPK activation has not been demonstrated.
We show that loss of MEKK1 results in temperature-sensitive and tissue-specific cell death and H 2 O 2 accumulation
that are partly dependent on both RAR1, a key component in resistance (R) protein function, and SID2, an isochorismate
synthase required for salicylic acid production upon pathogen infection. MEKK1 is not required for flg22-triggered
activation of MPK3 and MPK6. Instead, MEKK1 is essential for activation of MPK4, which is a negative regulator
of defense responses (6). These data suggest that MEKK1 plays an important role in negative control of defense
responses.
1. Asai et al., Nature 415:977, (2002)
2. Mizoguchi et al., FEBS Lett. 437:56, (1998)
3. Ichimura et al., Biochem. Biophys. Res. Commun. 253:532 (1998)
4. Teige et al., Mol. Cell 15:141 (2004)
5. Droillard et al., FEBS Lett. 574:42 (2004)
6. Peterson et al., Cell 103:1111 (2000)
278 Application of Laser Microdissection to the study of a powdery mildew-Arabidopsis
pathosystem
Noriko Inada 1 , Mary Wildermuth 2
1
Nara Institute of Science and Technology, 2 University of California, Berkeley
The powdery mildew Golovinomyces orontii (formerly named Erysiphe orontii) is an obligate biotrophic fungus that
infects epidermal cells of Arabidopsis thaliana leaves. When powdery mildew conidia are inoculated on a leaf surface,
conidial germination occurs at 2 hrs after inoculation (hpi), followed by appresorial formation and penetration at 5 hpi, and
haustorial formation at 24 hpi. Later, fungi grow mycelium on the leaf surface and develop conidiophores to produce more
conidia. Most research elucidating the powdery mildew-Arabidopsis interaction has focused on later stages of infection
when powdery mildew is visible to the naked eye (~5-7 dpi) due to the time-intensive nature of microscopic evaluation
and lack of markers for early infection stages. In addition, conventional whole leaf analysis does not differentiate events
in infected cells from those of neighboring uninfected cells. With this in mind, we are employing Laser Microdissection
(LMD) to analyze populations of infected epidermal cells and uninfected epidermal and mesophyll cells separately. This
powerful method has been utilized for animal research, yet is still new to plant biology. As the first step, we optimized
fixation and paraffin embedding methods suitable for fragile mature Arabidopsis leaves. High quality histology as well
as RNA were obtained from paraffin sections prepared by the microwave method. RT-PCR analysis clearly exhibited
the specificity of laser dissected cells. We then optimized method for RNA extraction and amplification for the use with
Affymetrix Arabidopsis GeneChip. We are currently in the process of microarray analysis for infected and uninfected
cells at 5 dpi. The temporally and spatially resolved global expression data generated through this work will facilitate
detailed mechanistic understanding of the powdery mildew-Arabidopsis interaction and derivation of the regulatory
circuitry associated with this interaction.