Annual Report 2006

analogous to vertebrates immunity system
based on the recognition of pathogens specific
molecule (antigen) with the highly variable
antibody: immunoglobulins. However, although
plants lack special diversifying system of the R-
genes as the case of vertebrate globulin
molecules, we have shown that the genome
regions around the R-genes are high in the
mutation frequencies (2004 Annual Report),
especially in the base substitution rate. In order
to clarify whether this accumulation of
variability around the R-genes are due to the
simple selective accumulation, or reflecting the
higher mutation rate in this region, we have
assessed the real-time mutation (basesubstitution)
rate in the genome
and checked the correlation of this with the
distance from the R-genes. β-glucuronidase
(GUS) genes with a stop codon mutation was
transformed into , and the
transformants were checked for their insertion
sites in the genome. As shown in the Fig. 5, the
somatic mutation during the individual
development was assessed by the development
of revertant of the GUS genes as the blue spot
of digested X-Gluc. Some correlation of higher
substitution rate with the R-gene was
suggested, and more confirmation is now being
done. If this was confirmed, it will become the
first case of verification of the plant R-gene
mutation development system.
Catalytic activation of plant
MAPK phosphatase NtMKP1 by
its physiological substrate SIPK
In plants, two MAPKs, wound-induced
protein kinase (WIPK) and salicylic acidinduced
protein kinase (SIPK), are key
molecules of signal transduction upon wound
and disease responses. The activity of MAPKs
is strictly regulated via phosphorylation by an
upstream MAPK kinase. Conversely, MAPKs
are dephosphorylated and inactivated by
protein phosphatases. Previously, we identified
NtMKP1 (MAPK phosphatase) as a novel
calmodulin (CaM)-binding protein. Here, we
investigated the interaction between NtMKP1
and substrate MAPKs or CaMs. NtMKP1
inactivated SIPK through dephosphorylation.
CaM interacted with NtMKP1, but did not
activate its phosphatase activity. NtMKP1 is
composed of four domains: a dual-specificity
phosphatase catalytic domain, a gelsolin
homology domain, a CaM-binding domain, and
C-terminal domain. Deletion analysis revealed
that the N-terminal non-catalytic region of
NtMKP1 interacted with SIPK and was
essential for inactivating SIPK, whereas the
CaM-binding and C-terminal domains were
dispensable. Moreover, the phosphatase activity
of NtMKP1 was increased strongly by the
binding of SIPK, but weakly by WIPK, another
MAPK. The strong activation of NtMKP1
Fig. 5
Development of GUS blue spots revealing mutations in
the genome
This spots frequency correlated with the GUS
localization in the genome suggests the presence of
hot spots of mutation in the plant genome, related to
the plant disease resistance genes to recognize the
corresponding pathogens avirulence molecules.
Fig 6
Transient expression of NtMKP1 suppresses MEK DD -
induced cell death
Expression of constitutively active MAPK kinase
(MEK DD )in induced activation
of MAPK (WIPK and SIPK) and cell death.
Simultaneous expression of NtMKP1 compromised the
constitutively active MAPK kinase-induced responses.
NtMKP1 and MEK DD were transiently expressed in
by agroinfiltration. The photograph was
taken at 3 days after agroinfiltration.