15th International Conference on Arabidopsis Research - TAIR

T05-085
Prediction of multiple Arabidopsis targets for the
Pseudomonas effector protease AvrRpt2
Stephen Chisholm(1, 3), Douglas Dahlbeck(1, 3), Nandini Krishnamurthy(2, 3),
Kimmen Sjolander(2, 3), Brian Staskawicz(1, 3)
1-Department of Plant and Microbial Biology
2-Department of Bioengineering
3-University of California, Berkeley
During infection, the bacterium Pseudomonas syringae secretes effector
proteins such as AvrRpt2 into Arabidopsis thaliana cells. Following delivery
into the plant cell, AvrRpt2 is N-terminally processed to its mature form,
which has cysteine protease activity that causes elimination of the Arabidopsis
RIN4 protein. Elimination of RIN4 activates the disease resistance protein
RPS2, resulting in localized cell death, systemic resistance and decreased
pathogen proliferation. Mutation of predicted catalytic triad residues of
AvrRpt2 prevents AvrRpt2 processing, RIN4 elimination and RPS2-mediated
pathogen recognition.
In plants lacking RPS2, AvrRpt2 contributes to pathogen virulence. Interestingly,
AvrRpt2 increases virulence even in the absence of RIN4, suggesting
that elimination of RIN4 is not the sole function of AvrRpt2 and that additional
Arabidopsis proteins are targets of its protease activity. To facilitate identification
of AvrRpt2 targets, the cleavage recognition sequence for AvrRpt2 was
determined. Variants of this seven amino acid sequence span the AvrRpt2
processing site and occur twice in RIN4. Mutation of these sites in AvrRpt2
and RIN4 prevent processing and elimination, respectively. Furthermore, a
synthetic substrate containing these sequences is cleaved by AvrRpt2, demonstrating
that seven amino acids are sufficient to elicit AvrRpt2-dependent
cleavage. Based on these sequences, hidden Markov models were developed
to identify Arabidopsis proteins containing putative AvrRpt2 cleavage sites.
The potential of these proteins to serve as substrates for AvrRpt2 protease
activity will be presented.
15 th <strong>International</strong> <strong>Conference</strong> on Arabidopsis Research 2004 · Berlin
T05-086
Functional analysis and expression studies of the
flagellin receptor FLS2
Silke Robatzek(1), Delphine Chinchilla(1), Zsuzsa Bauer(1), Cyril Zipfel(1), Gernot
Kunze(1), Pascal Bittel(1), Anne Caniard(1), Georg Felix(1), Thomas Boller(1)
1-Botanical Insitute, University Basel, Hebelstr. 1, 4056 Basel, Switzerland
Innate immunity is based on the recognition of so-called pathogen-associated
molecular patterns (PAMPs). One prominent PAMP in plants and animals
is flagellin, the major component of the bacterial motility organ. Flagellin
signalling in Arabidopsis is initiated by the perception of its most conserved
domain, represented by the flg22 peptide. This leads to the activation of a
MAP kinase cascade, the induction of numerous defence-related genes, and
induced resistance to virulent bacteria. The FLS2 gene, encoding the receptor-like
kinase FLS2, is essential for flagellin signalling, since fls2 mutants are
insensitive to flg22.
Here, we demonstrate that FLS2 functions as the flagellin receptor. Like the
previously identified flg22 binding site, FLS2 is found to be a glycosylated
protein. Cross-linking experiments with radioactive labelled flg22 reveal a
band of ~160 kDa, which co-migrates with a band detected by antibodies
against FLS2. Moreover, in plants expressing a FLS2-GFP fusion protein, both
the cross-linked and the immuno-detected bands are shifted to ~190 kDa.
In previous work we have shown that the protein kinase activity of FLS2 is
not only required for signal transmission, but also for flg22 binding. Here, we
focus on several conserved serine and threonine residues within the protein
kinase domain of FLS2. We have transformed fls2 mutant plants with FLS2
constructs, in which these residues are substituted. All three FLS2 mutations
in one of the three conserved threonines are able to restore binding of flg22,
but not the early and late responses to flg22. Thus, phosphorylation of these
residues appears to be critical for signal transmission but not for binding.
We have generated transgenic fls2 mutant plants expressing an FLS2-GFP
fusion under the control of its native promoter, which re-gain full responsiveness
to flg22. Using confocal microscopy, we show that FLS2-GFP expression
in leaves occurs in mesophyll and epidermal cells including stomata,
the possible entry sites for invading bacteria. Furthermore, the fluorescence
signal is also present in roots, stems and flowers, and it is always clearly
localized to the plasma membrane. Ongoing studies focus on the localization
of the fluorescence signal after treatment of the transgenic plants with flg22.
T05 Interaction with the Environment 2 (Biotic)