Hann Ling Wong

Hd3a Protein Is a Mobile Flowering Signal in Rice

Florigen, the mobile signal that moves from an induced leaf to the shoot apex and causes flowering, has eluded identification since it was first proposed 70 years ago. Understanding the nature of the mobile flowering signal would provide a key insight into the molecular mechanism of floral induction. Recent studies suggest that the Arabidopsis FLOWERING LOCUS T (FT) gene is a candidate for encoding florigen. We show that the protein encoded by Hd3a, a rice ortholog of FT, moves from the leaf to the shoot apical meristem and induces flowering in rice. These results suggest that the Hd3a protein may be the rice florigen.

Regulation of Rice NADPH Oxidase by Binding of Rac GTPase to Its N-Terminal Extension

Reactive oxygen species (ROS) produced by NADPH oxidase play critical roles in various cellular activities, including plant innate immunity response. In contrast with the large multiprotein NADPH oxidase complex of phagocytes, in plants, only the homologs of the catalytic subunit gp91phox and the cytosolic regulator small GTPase Rac are found. Plant homologs of the gp91phox subunit are known as Rboh (for respiratory burst oxidase homolog). Although numerous Rboh have been isolated in plants, the regulation of enzymatic activity remains unknown. All rboh genes identified to date possess a conserved N-terminal extension that contains two Ca2+ binding EF-hand motifs. Previously, we ascertained that a small GTPase Rac (Os Rac1) enhanced pathogen-associated molecular pattern–induced ROS production and resistance to pathogens in rice (Oryza sativa). In this study, using yeast two-hybrid assay, we found that interaction between Rac GTPases and the N-terminal extension is ubiquitous and that a substantial part of the N-terminal region of Rboh, including the two EF-hand motifs, is required for the interaction. The direct Rac–Rboh interaction was supported by further studies using in vitro pull-down assay, a nuclear magnetic resonance titration experiment, and in vivo fluorescence resonance energy transfer (FRET) microscopy. The FRET analysis also suggests that cytosolic Ca2+ concentration may regulate Rac–Rboh interaction in a dynamic manner. Furthermore, transient coexpression of Os Rac1 and rbohB enhanced ROS production in Nicotiana benthamiana, suggesting that direct Rac–Rboh interaction may activate NADPH oxidase activity in plants. Taken together, the results suggest that cytosolic Ca2+ concentration may modulate NADPH oxidase activity by regulating the interaction between Rac GTPase and Rboh.

Down-regulation of metallothionein, a reactive oxygen scavenger, by the small GTPase OsRac1 in rice

Metallothioneins are small, ubiquitous Cys-rich proteins known to be involved in reactive oxygen species (ROS) scavenging and metal homeostasis. We found that the expression of a metallothionein gene (OsMT2b) was synergically down-regulated by OsRac1 and rice (Oryza sativa) blast-derived elicitors. Transgenic plants overexpressing OsMT2b showed increased susceptibility to bacterial blight and blast fungus. OsMT2b-overexpressing cells showed reduced elicitor-induced hydrogen peroxide production. In contrast, homozygous OsMT2b::Tos17-inserted mutant and OsMT2b-RNAi-silenced transgenic cells showed significantly higher elicitor-induced hydrogen peroxide production than the wild-type cells. In vitro assay showed that recombinant OsMT2b protein possessed superoxide- and hydroxyl radical-scavenging activities. Taken together, these results showed that OsMT2b is an ROS scavenger and its expression is down-regulated by OsRac1, thus potentiating ROS, which function as signals in resistance response. The results suggest that OsRac1 plays a dual role as an inducer of ROS production and a suppressor of ROS scavenging.

RACK1 Functions in Rice Innate Immunity by Interacting with the Rac1 Immune Complex

A small GTPase, Rac1, plays a key role in rice (Oryza sativa) innate immunity as part of a complex of regulatory proteins. Here, we used affinity column chromatography to identify rice RACK1 (for Receptor for Activated C-Kinase 1) as an interactor with Rac1. RACK1 functions in various mammalian signaling pathways and is involved in hormone signaling and development in plants. Rice contains two RACK1 genes, RACK1A and RACK1B, and the RACK1A protein interacts with the GTP form of Rac1. Rac1 positively regulates RACK1A at both the transcriptional and posttranscriptional levels. RACK1A transcription was also induced by a fungal elicitor and by abscisic acid, jasmonate, and auxin. Analysis of transgenic rice plants and cell cultures indicates that RACK1A plays a role in the production of reactive oxygen species (ROS) and in resistance against rice blast infection. Overexpression of RACK1A enhances ROS production in rice seedlings. RACK1A was shown to interact with the N terminus of NADPH oxidase, RAR1, and SGT1, key regulators of plant disease resistance. These results suggest that RACK1A functions in rice innate immunity by interacting with multiple proteins in the Rac1 immune complex.

Sekiguchi lesion gene encodes a cytochrome p450 monooxygenase that catalyzes conversion of tryptamine to serotonin in rice

Serotonin is a well known neurotransmitter in mammals and plays an important role in various mental functions in humans. In plants, the serotonin biosynthesis pathway and its function are not well understood. The rice sekiguchi lesion (sl) mutants accumulate tryptamine, a candidate substrate for serotonin biosynthesis. We isolated the SL gene by map-based cloning and found that it encodes CYP71P1 in a cytochrome P450 monooxygenase family. A recombinant SL protein exhibited tryptamine 5-hydroxylase enzyme activity and catalyzed the conversion of tryptamine to serotonin. This pathway is novel and has not been reported in mammals. Expression of SL was induced by the N-acetylchitooligosaccharide (chitin) elicitor and by infection with Magnaporthe grisea, a causal agent for rice blast disease. Exogenously applied serotonin induced defense gene expression and cell death in rice suspension cultures and increased resistance to rice blast infection in plants. We also found that serotonin-induced defense gene expression is mediated by the RacGTPase pathway and by the Gα subunit of the heterotrimeric G protein. These results suggest that serotonin plays an important role in rice innate immunity.

Structure of the N-terminal Regulatory Domain of a Plant NADPH Oxidase and Its Functional Implications

Plant NADPH oxidases (Rboh, for respiratory burst oxidase homolog) produce reactive oxygen species that are key regulators of various cellular events including plant innate immunity. Rbohs possess a highly conserved cytoplasmic N-terminal region containing two EF-hand motifs that regulate Rboh activity. Rice (Oryza sativa) RbohB (OsRbohB) is regulated by the direct binding of a small GTPase (Rac1) to this regulatory region as well as by Ca2+ binding to the EF-hands. Here, we present the atomic structure of the N-terminal region of OsRbohB. The structure reveals that OsRbohB forms a unique dimer stabilized by swapping the EF-hand motifs. We identified two additional EF-hand-like motifs that were not predicted from sequence data so far. These EF-hand-like motifs together with the swapped EF-hands form a structure similar to that found in calcineurin B. We observed conformational changes mediated by Ca2+ binding to only one EF-hand. Structure-based in vitro pulldown assays and NMR titration experiments defined the OsRac1 binding interface within the coiled-coil region created by swapping the EF-hands. In addition, we demonstrate a direct intramolecular interaction between the N and C terminus, and that the complete N-terminal cytoplasmic region is required for this interaction. The structural features and intramolecular interactions characterized here might be common elements shared by Rbohs that contribute to the regulation of reactive oxygen species production.

Activation of a Rac GTPase by the NLR Family Disease Resistance Protein Pit Plays a Critical Role in Rice Innate Immunity

The nucleotide-binding domain and leucine-rich repeat-containing (NLR) family proteins recognize pathogen-derived molecules and trigger immune responses in both plants and animals. In plants, the direct or indirect recognition of specific pathogen effectors by NLRs culminates in a hypersensitive response (HR) and the production of reactive oxygen species (ROS), key components of the plant defense response. However, the molecules activated by NLRs and how they induce immune responses are largely unknown. We found that the rice GTPase OsRac1 at the plasma membrane interacts directly with Pit, an NLR protein that confers resistance to the rice blast fungus. OsRac1 contributes to Pit-mediated ROS production as well as the HR and is required for Pit-mediated disease resistance in rice. Furthermore, the active form of Pit induces the activation of OsRac1 at the plasma membrane. Thus, OsRac1 is activated by Pit during pathogen attack and plays a critical role in Pit-mediated immunity in rice.

Analysis of the Rac/Rop Small GTPase Family in Rice: Expression, Subcellular Localization and Role in Disease Resistance

Plant-specific Rac/Rop small GTPases function as molecular switches for numerous signal transduction events, including defense responses. To understand the function of each of the seven Rac/Rop family members in rice, we studied the tissue-specific expression patterns of Rac/Rop genes by semi-quantitative reverse transcription-PCR (RT-PCR), and also Rac/Rop subcellular localization using green fluorescent protein (GFP) fusion proteins in transient expression systems. We also investigated the roles of these genes in disease resistance by testing single Rac/Rop-RNAi (RNA interference) plants against the rice blast pathogen Magnaporthe grisea. Our studies show that expression of OsRac2, 6 and 7 is very low in leaf blades, and reveal a strong correlation between the number of lysine and/or arginine (KR) residues in the polybasic region of Rac/Rop GTPases and their subcellular distribution in vivo. Infection assays showed that OsRac1 is a positive regulator of blast resistance, confirming previous observations, whereas OsRac4 and OsRac5 are negative regulators of blast resistance. OsRac6 may make minor contributions to disease resistance, while OsRac3 and OsRac7 are probably not involved in defense. Therefore, our study suggests that the rice Rac/Rop family plays multiple roles in diverse cellular activities and has both positive and negative functions in disease resistance.

RAV-Like1 Maintains Brassinosteroid Homeostasis via the Coordinated Activation of BRI1 and Biosynthetic Genes in Rice

Brassinosteroid (BR) homeostasis is established by the regulatory circuit between receptor BRI1-mediated signaling and BR synthesis. RAVL1 modulates the strength of the circuit by activating expression of both BRI1 and synthetic genes and is necessary for feedback responses to BR levels. Temporal and spatial variation in the levels of and sensitivity to hormones are essential for the development of higher organisms. Traditionally, end-product feedback regulation has been considered as the key mechanism for the achievement of cellular homeostasis. Brassinosteroids (BRs) are plant steroid hormones that are perceived by the cell surface receptor kinase Brassinosteroid Insensitive1. Binding of these hormones to the receptor activates BR signaling and eventually suppresses BR synthesis. This report shows that RAVL1 regulates the expression of the BR receptor. Furthermore, RAVL1 is also required for the expression of the BR biosynthetic genes D2, D11, and BRD1 that are subject to BR negative feedback. Activation by RAVL1 was coordinated via E-box cis-elements in the promoters of the receptor and biosynthetic genes. Also, RAVL1 is necessary for the response of these genes to changes in cellular BR homeostasis. Genetic evidence is presented to strengthen the observation that the primary action of RAVL1 mediates the expression of genes involved in BR signaling and biosynthesis. This study thus describes a regulatory circuit modulating the homeostasis of BR in which RAVL1 ensures the basal activity of both the signaling and the biosynthetic pathways.

The bHLH Rac Immunity1 (RAI1) Is Activated by OsRac1 via OsMAPK3 and OsMAPK6 in Rice Immunity.

The Rac/Rop GTPase OsRac1 plays an essential role in rice immunity. However, the regulatory genes acting downstream of OsRac1 are largely unknown. We focused on the RAI1 gene, which is up-regulated in suspension cells expressing a constitutively active form of OsRac1. RAI1 encodes a putative basic helix-loop-helix transcription factor. A microarray analysis of cells transformed with an inducible RAI1 construct showed increased expression of PAL1 and OsWRKY19 genes after induction, suggesting that these genes are regulated by RAI1. This was confirmed using RAI1 T-DNA activation-tagged and RNA interference lines. The PAL1 and OsWRKY19 genes were also up-regulated by sphingolipid and chitin elicitors, and the RAI1 activation-tagged plants had increased resistance to a rice blast fungus. These results indicated that RAI1 is involved in defense responses in rice. RAI1 interacted with OsMAPK3 and OsMAPK6 proteins in vivo and in vitro. Also, RAI1 was phosphorylated by OsMAPK3/6 and OsMKK4-dd in vitro. Overexpression of OsMAPK6 and/or OsMAPK3 together with OsMKK4-dd increased PAL1 and OsWRKY19 expression in rice protoplasts. Therefore, the regulation of PAL1 and OsWRKY19 expression by RAI1 could be controlled via an OsMKK4-OsMAPK3/6 cascade. Co-immunoprecipitation assays indicated that OsMAPK3 and OsRac1 occur in the same complex as OsMAPK6. Taken together, our results indicate that RAI1 could be regulated by OsRac1 through an OsMAPK3/6 cascade. In this study, we have identified RAI1 as the first transcription factor acting downstream of OsRac1. This work will help us to understand the immune system regulated by OsRac1 in rice and its orthologs in other plant species.