In vivo monitoring of plant small GTPase activation using a Förster resonance energy transfer biosensor

Wong, Hann Ling; Akamatsu, Akira; Wang, Qiong; Higuchi, Makie; Matsuda, Tomonori; Okuda, Jun; Kosami, Ken ichi; Inada, Noriko; Kawasaki, Tsutomu; Kaneko-Kawano, Takako; Nagawa, Shingo; Li, Tan; Kawano, Yoji; Shimamoto, Ko

doi:10.1186/s13007-018-0325-4

Cited by 16 publications

(20 citation statements)

References 31 publications

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“…4A), indicating that OsSPK1 possesses GEF activity for OsRac1 in vitro. To monitor in vivo activation of OsRac1 by OsSPK1, we employed a FRET sensor called Ras and interacting protein chimeric unit (Raichu)-OsRac1 (37,45). In this sensor, intramolecular binding of the active GTP-OsRac1 to the Cdc42/Rac interactive binding domain brings CFP closer to Venus, enabling FRET from CFP to Venus when OsRac1 is activated (Fig.…”

Section: Resultsmentioning

confidence: 99%

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Resistance protein Pit interacts with the GEF OsSPK1 to activate OsRac1 and trigger rice immunity

Wang

Ishikawa

et al. 2018

Proc. Natl. Acad. Sci. U.S.A.

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Resistance (R) genes encode intracellular nucleotide-binding/leucine-rich repeat-containing (NLR) family proteins that serve as critical plant immune receptors to induce effector-triggered immunity (ETI). NLR proteins possess a tripartite domain architecture consisting of an N-terminal variable region, a central nucleotide-binding domain, and a C-terminal leucine-rich repeat. N-terminal coiled-coil (CC) or Toll-interleukin 1 receptor (TIR) domains of R proteins appear to serve as platforms to trigger immune responses, because overexpression of the CC or TIR domain of some R proteins is sufficient to induce an immune response. Because direct downstream signaling molecules of R proteins remain obscure, the molecular mechanisms by which R proteins regulate downstream signaling are largely unknown. We reported previously that a rice R protein named Pit triggers ETI through a small GTPase, OsRac1, although how Pit activates OsRac1 is unclear. Here, we identified OsSPK1, a DOCK family guanine nucleotide exchange factor, as an interactor of Pit and activator for OsRac1. OsSPK1 contributes to signaling by two disease-resistance genes, Pit and Pia, against the rice blast fungus Magnaporthe oryzae and facilitates OsRac1 activation in vitro and in vivo. The CC domain of Pit is required for its binding to OsSPK1, OsRac1 activation, and the induction of cell death. Overall, we conclude that OsSPK1 is a direct and key signaling target of Pit-mediated immunity. Our results shed light on how R proteins trigger ETI through direct downstream molecules.

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Section: Resultsmentioning

confidence: 99%

“…FRET Analysis. To analyze the activation of OsRac1 by OsSPK1 in vivo, we employed the Raichu intramolecular FRET system, as described previously (37,45). Rice protoplasts were transfected with Raichu-OsRac1 and OsSPK1 (amino acids 1002-1835) or GUS vectors by the PEG method.…”

Section: Raichu-osrac1mentioning

confidence: 99%

Resistance protein Pit interacts with the GEF OsSPK1 to activate OsRac1 and trigger rice immunity

Wang

Ishikawa

et al. 2018

Proc. Natl. Acad. Sci. U.S.A.

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“…It will be interesting to test whether active and inactive forms of PpROP1 are in fact spatially segregated within the dome-shaped PpROP1 labeled plasma membrane region at tip of apical protonemal cells. To this end, the intracellular distributions in these cells of P. patens ROP-GAP homologs as well as of markers specific for active PpROP1 [fluorescent CRIB-domain fusion proteins (Hwang et al, 2005) or FRET sensors (Wong et al, 2018)] can be investigated using the tools and methods developed in the course of the study. In any case, a possible function of PpROP-GEF4 in PpROP1 activation in apical protonemal cells is further supported also by the observations that both proteins are highly and preferentially expressed in these cells as determined based on qPCR analysis and confocal imaging of “knock-in” lines, and interact with each other in yeast two-hybrid assays.…”

Section: Discussionmentioning

confidence: 99%

Analysis of the Localization of Fluorescent PpROP1 and PpROP-GEF4 Fusion Proteins in Moss Protonemata Based on Genomic “Knock-In” and Estradiol-Titratable Expression

et al. 2019

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Tip growth of pollen tubes, root hairs, and apical cells of moss protonemata is controlled by ROP (Rho of plants) GTPases, which were shown to accumulate at the apical plasma membrane of these cells. However, most ROP localization patterns reported in the literature are based on fluorescent protein tagging and need to be interpreted with caution, as ROP fusion proteins were generally overexpressed at undefined levels, in many cases without assessing effects on tip growth. ROP-GEFs, important regulators of ROP activity, were also described to accumulate at the apical plasma membrane during tip growth. However, to date only the localization of fluorescent ROP-GEF fusion proteins strongly overexpressed using highly active promoters have been investigated. Here, the intracellular distributions of fluorescent PpROP1 and PpROP-GEF4 fusion proteins expressed at essentially endogenous levels in apical cells of Physcomitrella patens “knock-in” protonemata were analyzed. Whereas PpROP-GEF4 was found to associate with a small apical plasma membrane domain, PpROP1 expression was below the detection limit. Estradiol-titratable expression of a fluorescent PpROP1 fusion protein at the lowest detectable level, at which plant development was only marginally affected, was therefore employed to show that PpROP1 also accumulates at the apical plasma membrane, although within a substantially larger domain. Interestingly, RNA-Seq data indicated that the majority of all genes active in protonemata are expressed at lower levels than PpROP1 , suggesting that estradiol-titratable expression may represent an important alternative to “knock-in” based analysis of the intracellular distribution of fluorescent fusion proteins in protonemal cells.

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“…The final constructs were transformed into Nipponbare calli via Agrobacterium strain EHA105. Rice suspension cells derived from Nipponbare calli were grown in R2 liquid medium at 30°C and the medium was changed once a week (Hayakawa et al, 1992, Wong et al, 2018).…”

Section: Methodsmentioning

confidence: 99%

The secreted peptide IRP functions as a DAMP in rice immunity

Wang

Jia

Guo

et al. 2021

Preprint

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Small signaling peptides play important roles in various plant processes, but information regarding their involvement in plant immunity is limited. We previously identified a novel small secreted protein in rice, named immune response peptide (IRP) by the integrated multi-omics analyses. Here, we studied IRP functions in rice immunity. Rice plants overexpressing IRP enhanced resistance to the blast fungus. Application of the IRP peptide to rice suspension cells triggered the expression of IRP itself and defense gene PAL1. RNA-seq results revealed that 84% of genes upregulated by IRP peptide were also induced by chitin, including 13 OsWRKY transcription factors, indicating that IRP and chitin share the similar signaling pathway. Co-treatment with chitin and IRP elevated the expression level of PAL1 and OsWRKYs in an additive manner. When results of IRP and PAL1 expression and MAPK activation by IRP were compared with those by chitin, IRP had a stronger effect on MAPK activation rather than IRP and PAL1 expression. Collectively, our findings indicate that IRP functions as a damage-associated molecular pattern (DAMP) in rice immunity, regulating MAPKs and OsWRKYs to amplify chitin signaling, and provide new insights into how PAMPs and DAMPs cooperatively regulate rice immunity.

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In vivo monitoring of plant small GTPase activation using a Förster resonance energy transfer biosensor

Cited by 16 publications

References 31 publications

Resistance protein Pit interacts with the GEF OsSPK1 to activate OsRac1 and trigger rice immunity

Resistance protein Pit interacts with the GEF OsSPK1 to activate OsRac1 and trigger rice immunity

Analysis of the Localization of Fluorescent PpROP1 and PpROP-GEF4 Fusion Proteins in Moss Protonemata Based on Genomic “Knock-In” and Estradiol-Titratable Expression

The secreted peptide IRP functions as a DAMP in rice immunity

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