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Rho family GTPase-dependent immunity in plants and animals.

Kawano Y, Kaneko-Kawano T, Shimamoto K - Front Plant Sci (2014)

Bottom Line: Rac/Rop GTPases trigger various immune responses, thereby resulting in enhanced disease resistance to pathogens.In this review, we highlight recent studies that have contributed to our current understanding of the Rac/Rop family GTPases and the upstream and downstream proteins involved in plant immunity.We also compare the features of effector-triggered immunity between plants and animals, and discuss the in vivo monitoring of Rac/Rop activation.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Plant Molecular Genetics, Nara Institute of Science and Technology Ikoma, Japan.

ABSTRACT
In plants, sophisticated forms of immune systems have developed to cope with a variety of pathogens. Accumulating evidence indicates that Rac (also known as Rop), a member of the Rho family of small GTPases, is a key regulator of immunity in plants and animals. Like other small GTPases, Rac/Rop GTPases function as a molecular switch downstream of immune receptors by cycling between GDP-bound inactive and GTP-bound active forms in cells. Rac/Rop GTPases trigger various immune responses, thereby resulting in enhanced disease resistance to pathogens. In this review, we highlight recent studies that have contributed to our current understanding of the Rac/Rop family GTPases and the upstream and downstream proteins involved in plant immunity. We also compare the features of effector-triggered immunity between plants and animals, and discuss the in vivo monitoring of Rac/Rop activation.

No MeSH data available.


In vivo monitoring of Rac/Rop activation using a GST-CRIB pull-down assay. The CRIB domain of PAK has a high affinity for the active GTP-bound form of Rac/Rop. PAK-CRIB-binding to Rac/Rop suppresses the intrinsic and catalytic rates of GTP hydrolysis of Rac/Rop that make it possible to purify the active constitutively active (CA) form but not the inactive dominant negative (DN) form of OsRac1 from cell lysates (Kawano et al., 2010a). A figure from Kawano et al. (2010a) was adapted for Figure 6.
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Figure 6: In vivo monitoring of Rac/Rop activation using a GST-CRIB pull-down assay. The CRIB domain of PAK has a high affinity for the active GTP-bound form of Rac/Rop. PAK-CRIB-binding to Rac/Rop suppresses the intrinsic and catalytic rates of GTP hydrolysis of Rac/Rop that make it possible to purify the active constitutively active (CA) form but not the inactive dominant negative (DN) form of OsRac1 from cell lysates (Kawano et al., 2010a). A figure from Kawano et al. (2010a) was adapted for Figure 6.

Mentions: Given that Rac/Rop is a master regulator controlling plant immunity, monitoring its activation within plant cells is believed to be the next key step in understanding plant immunity. Traditionally, small GTPase activities are measured using in vivo labeling of cells with of inorganic [32P] phosphate followed by isolation of the GTPase and thin-layer chromatography of bound guanine nucleotides. This method provides quantitative data for GDP and GTP levels on small GTPases but is a time-consuming procedure that requires large amounts of radioisotopes. Currently, we are able to use two alternative non-radioactive techniques, a PAK-CRIB pull-down assay and a Raichu-Förster resonance energy transfer (FRET) sensor, to monitor the in vivo activation of Rac/Rop (Figures 6 and 7; Sander et al., 1998; Mochizuki et al., 2001; Tao et al., 2002; Kawano et al., 2010a). These methods exploit the selective interaction of the CRIB of the Rac-effector PAK1 in animals. Since the CRIB-domain of PAK has a high affinity for the active GTP-bound form of Rac/Rop and PAK-CRIB binding results in a significantly reduced intrinsic GTPase activity of Rac/Rop, these factors result in an ideal tool for affinity purification of active GTP-bound forms of Rac/Rop from crude cell lysates. Recombinant GST-tagged PAK-CRIB protein is currently available from several manufacturers. GST-tagged PAK-CRIB allows one to “pull-down” the PAK-CRIB/GTP.Rac/Rop complex with glutathione affinity beads (Figure 6). Therefore, the assay provides a simple means of quantifying Rac/Rop activation in cells. The amount of activated Rac is determined by immunoblotting. This approach has greatly accelerated and, thus, simplified the semi-quantitative measurement of Rac activity in plants and animals (Sander et al., 1998; Tao et al., 2002; Xu et al., 2010).


Rho family GTPase-dependent immunity in plants and animals.

Kawano Y, Kaneko-Kawano T, Shimamoto K - Front Plant Sci (2014)

In vivo monitoring of Rac/Rop activation using a GST-CRIB pull-down assay. The CRIB domain of PAK has a high affinity for the active GTP-bound form of Rac/Rop. PAK-CRIB-binding to Rac/Rop suppresses the intrinsic and catalytic rates of GTP hydrolysis of Rac/Rop that make it possible to purify the active constitutively active (CA) form but not the inactive dominant negative (DN) form of OsRac1 from cell lysates (Kawano et al., 2010a). A figure from Kawano et al. (2010a) was adapted for Figure 6.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Figure 6: In vivo monitoring of Rac/Rop activation using a GST-CRIB pull-down assay. The CRIB domain of PAK has a high affinity for the active GTP-bound form of Rac/Rop. PAK-CRIB-binding to Rac/Rop suppresses the intrinsic and catalytic rates of GTP hydrolysis of Rac/Rop that make it possible to purify the active constitutively active (CA) form but not the inactive dominant negative (DN) form of OsRac1 from cell lysates (Kawano et al., 2010a). A figure from Kawano et al. (2010a) was adapted for Figure 6.
Mentions: Given that Rac/Rop is a master regulator controlling plant immunity, monitoring its activation within plant cells is believed to be the next key step in understanding plant immunity. Traditionally, small GTPase activities are measured using in vivo labeling of cells with of inorganic [32P] phosphate followed by isolation of the GTPase and thin-layer chromatography of bound guanine nucleotides. This method provides quantitative data for GDP and GTP levels on small GTPases but is a time-consuming procedure that requires large amounts of radioisotopes. Currently, we are able to use two alternative non-radioactive techniques, a PAK-CRIB pull-down assay and a Raichu-Förster resonance energy transfer (FRET) sensor, to monitor the in vivo activation of Rac/Rop (Figures 6 and 7; Sander et al., 1998; Mochizuki et al., 2001; Tao et al., 2002; Kawano et al., 2010a). These methods exploit the selective interaction of the CRIB of the Rac-effector PAK1 in animals. Since the CRIB-domain of PAK has a high affinity for the active GTP-bound form of Rac/Rop and PAK-CRIB binding results in a significantly reduced intrinsic GTPase activity of Rac/Rop, these factors result in an ideal tool for affinity purification of active GTP-bound forms of Rac/Rop from crude cell lysates. Recombinant GST-tagged PAK-CRIB protein is currently available from several manufacturers. GST-tagged PAK-CRIB allows one to “pull-down” the PAK-CRIB/GTP.Rac/Rop complex with glutathione affinity beads (Figure 6). Therefore, the assay provides a simple means of quantifying Rac/Rop activation in cells. The amount of activated Rac is determined by immunoblotting. This approach has greatly accelerated and, thus, simplified the semi-quantitative measurement of Rac activity in plants and animals (Sander et al., 1998; Tao et al., 2002; Xu et al., 2010).

Bottom Line: Rac/Rop GTPases trigger various immune responses, thereby resulting in enhanced disease resistance to pathogens.In this review, we highlight recent studies that have contributed to our current understanding of the Rac/Rop family GTPases and the upstream and downstream proteins involved in plant immunity.We also compare the features of effector-triggered immunity between plants and animals, and discuss the in vivo monitoring of Rac/Rop activation.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Plant Molecular Genetics, Nara Institute of Science and Technology Ikoma, Japan.

ABSTRACT
In plants, sophisticated forms of immune systems have developed to cope with a variety of pathogens. Accumulating evidence indicates that Rac (also known as Rop), a member of the Rho family of small GTPases, is a key regulator of immunity in plants and animals. Like other small GTPases, Rac/Rop GTPases function as a molecular switch downstream of immune receptors by cycling between GDP-bound inactive and GTP-bound active forms in cells. Rac/Rop GTPases trigger various immune responses, thereby resulting in enhanced disease resistance to pathogens. In this review, we highlight recent studies that have contributed to our current understanding of the Rac/Rop family GTPases and the upstream and downstream proteins involved in plant immunity. We also compare the features of effector-triggered immunity between plants and animals, and discuss the in vivo monitoring of Rac/Rop activation.

No MeSH data available.