Hau-Hsuan Hwang

Identification of Arabidopsis rat mutants

Limited knowledge currently exists regarding the roles of plant genes and proteins in the Agrobacterium tumefaciens-mediated transformation process. To understand the host contribution to transformation, we carried out root-based transformation assays to identify Arabidopsis mutants that are resistant to Agrobacterium transformation (rat mutants). To date, we have identified 126 rat mutants by screening libraries of T-DNA insertion mutants and by using various “reverse genetic” approaches. These mutants disrupt expression of genes of numerous categories, including chromatin structural and remodeling genes, and genes encoding proteins implicated in nuclear targeting, cell wall structure and metabolism, cytoskeleton structure and function, and signal transduction. Here, we present an update on the identification and characterization of these rat mutants.

Plant Proteins That Interact with VirB2, the Agrobacterium tumefaciens Pilin Protein, Mediate Plant Transformation

Agrobacterium tumefaciens uses a type IV secretion system (T4SS) to transfer T-DNA and virulence proteins to plants. The T4SS is composed of two major structural components: the T-pilus and a membrane-associated complex that is responsible for translocating substrates across both bacterial membranes. VirB2 protein is the major component of the T-pilus. We used the C-terminal–processed portion of VirB2 protein as a bait to screen an Arabidopsis thaliana cDNA library for proteins that interact with VirB2 in yeast. We identified three related plant proteins, VirB2-interacting protein (BTI) 1 (BTI1), BTI2, and BTI3 with unknown functions, and a membrane-associated GTPase, AtRAB8. The three BTI proteins also interacted with VirB2 in vitro. Preincubation of Agrobacterium with GST-BTI1 protein decreased the transformation efficiency of Arabidopsis suspension cells by Agrobacterium. Transgenic BTI and AtRAB8 antisense and RNA interference Arabidopsis plants are less susceptible to transformation by Agrobacterium than are wild-type plants. The level of BTI1 protein is transiently increased immediately after Agrobacterium infection. In addition, overexpression of BTI1 protein in transgenic Arabidopsis results in plants that are hypersusceptible to Agrobacterium-mediated transformation. Confocal microscopic data indicate that GFP-BTI proteins preferentially localize to the periphery of root cells in transgenic Arabidopsis plants, suggesting that BTI proteins may contact the Agrobacterium T-pilus. We propose that the three BTI proteins and AtRAB8 are involved in the initial interaction of Agrobacterium with plant cells.

Branchial FXYD protein expression in response to salinity change and its interaction with Na+/K+-ATPase of the euryhaline teleost Tetraodon nigroviridis.

SUMMARY Na+/K+-ATPase (NKA) is a ubiquitous membrane-bound protein crucial for teleost osmoregulation. The enzyme is composed of two essential subunits, a catalytic α subunit and a glycosylated β subunit which is responsible for membrane targeting of the enzyme. In mammals, seven FXYD members have been found. FXYD proteins have been identified as the regulatory protein of NKA in mammals and elasmobranchs, it is thus interesting to examine the expression and functions of FXYD protein in the euryhaline teleosts with salinity-dependent changes of gill NKA activity. The present study investigated the expression and distribution of the FXYD protein in gills of seawater (SW)- or freshwater (FW)-acclimated euryhaline pufferfish (Tetraodon nigroviridis). The full-length pufferfish FXYD gene (pFXYD) was confirmed by RT-PCR. pFXYD was found to be expressed in many organs including gills of both SW and FW pufferfish. pFXYD mRNA abundance in gills, determined by real-time PCR, was significantly higher in FW fish than in SW fish. An antiserum raised against a partial amino acid sequence of pFXYD was used for the immunoblots of gill homogenates and a major band at 13 kDa was detected. The relative amounts of pFXYD protein and mRNA in gills of SW and FW pufferfish were identical, but opposite to the expression levels of NKA. Immunofluorescent staining of frozen sections demonstrated that pFXYD was colocalized to NKA-immunoreactive cells in the gill filaments. In addition, interaction between pFXYD and NKA was demonstrated by co-immunoprecipitation. Taken together, salinity-dependent expression of pFXYD protein and NKA, as well as the evidence for their colocalization and interaction in pufferfish gills suggested that pFXYD regulates NKA activity in gills of euryhaline teleosts upon salinity challenge.

The Tzs Protein and Exogenous Cytokinin Affect Virulence Gene Expression and Bacterial Growth of Agrobacterium tumefaciens

The soil phytopathogen Agrobacterium tumefaciens causes crown gall disease in a wide range of plant species. The neoplastic growth at the infection sites is caused by transferring, integrating, and expressing transfer DNA (T-DNA) from A. tumefaciens into plant cells. A trans-zeatin synthesizing (tzs) gene is located in the nopaline-type tumor-inducing plasmid and causes trans-zeatin production in A. tumefaciens. Similar to known virulence (Vir) proteins that are induced by the vir gene inducer acetosyringone (AS) at acidic pH 5.5, Tzs protein is highly induced by AS under this growth condition but also constitutively expressed and moderately upregulated by AS at neutral pH 7.0. We found that the promoter activities and protein levels of several AS-induced vir genes increased in the tzs deletion mutant, a mutant with decreased tumorigenesis and transient transformation efficiencies, in Arabidopsis roots. During AS induction and infection of Arabidopsis roots, the tzs deletion mutant conferred impaired growth, which could be rescued by genetic complementation and supplementing exogenous cytokinin. Exogenous cytokinin also repressed vir promoter activities and Vir protein accumulation in both the wild-type and tzs mutant bacteria with AS induction. Thus, the tzs gene or its product, cytokinin, may be involved in regulating AS-induced vir gene expression and, therefore, affect bacterial growth and virulence during A. tumefaciens infection.

Editorial: "Agrobacterium biology and its application to transgenic plant production".

The extraordinary Agrobacterium research story started from the search for the causative agent of crown gall disease more than 100 years ago. Agrobacterium tumefaciens was first isolated from grapevine galls in 1897 and later isolated from Paris daisy in 1907 (Cavara, 1897a,b; Smith and Townsend, 1907). The Agrobacterium infection mechanism involves processing and transfer of a specific DNA fragment (the transferred-DNA, T-DNA) from a bacterial tumor-inducing (Ti) plasmid. Transfer to the plant occurs via a type IV secretion system (T4SS), after which T-DNA is integrated into the plant host genome (Gelvin, 2010; Lacroix and Citovsky, 2013). This interkingdom DNA transfer leads to overproduction of the plant hormones auxin and cytokinin, resulting in tumors. The interkingdom DNA transfer ability of Agrobacterium and the possibility to replace the oncogenes in the T-DNA with genes of interest has made Agrobacterium-mediated transformation the most popular technique to generate transgenic plants. This Research Topic provides a collection of reviews and original research articles on Agrobacterium genes involved in bacterial physiology/virulence and plant genes involved in transformation and defense against Agrobacterium. A review by Kado (2014) provides a historical overview of how A. tumefaciens was first established as the cause of crown gall disease. In this review, Kado highlights key early plant pathology and milestone molecular biology studies leading to the conclusion that the expression of oncogenes in native T-DNA is the cause of tumor growth in plants. With the solid foundation of these pioneering discoveries, A. tumefaciens evolved from a phytopathogen to a powerful genetic transformation tool for plant biology and biotechnology research. The first complete genome sequence of an Agrobacterium species (A. tumefaciens C58) was completed in 2001 (Goodner et al., 2001; Wood et al., 2001). The 5.67-megabase genome of this strain carries one circular chromosome, one linear chromosome, and two megaplasmids: the Ti plasmid pTiC58 and a second plasmid, pAtC58. In the review by Platt et al. (2014), the properties, ecology, evolution, and complex interactions of these two A. tumefaciens megaplasmids are discussed. The costs and benefits to A. tumefaciens strains carrying the Ti plasmid and/or the pAtC58 plasmid are discussed and presented from an ecological and evolutionary perspective. Modeling predictions are presented for the relative cost and benefits to A. tumefaciens strains harboring the Ti and/or the pAtC58 plasmids determined by environmental resources. Conjugation and amplification of the Ti plasmid are regulated by the TraI/TraR quorum-sensing (QS) system and conjugal opines. Lang and Faure (2014) review current knowledge of the genetic networks and molecular basis of the A. tumefaciens quorum sensing system. These authors also discuss the biological and ecological impact of the QS system on Ti plasmid conjugation, copy number, and interactions between Agrobacterium and host plants. During the initial interaction between Agrobacterium and plant cells, bacteria sense various plant-derived signals in the rhizosphere with the help of Ti plasmid-encoded virulence gene (vir gene) and chromosomal virulence gene (chv gene) products. The current knowledge of how A. tumefaciens senses and reacts to different plant-derived signals are summarized in the review article by Subramoni et al. (2014), which also discusses the mechanisms of how the plant hormones auxin, salicylic acid, and ethylene, affect bacterial virulence. Finally, this review discusses the complexity and intricacy of Agrobacterium signaling pathways and the underlying regulatory mechanisms during the initial host cell recognition to maximize subsequent successful infection. In the original research article by Lin et al. (2014), the mechanistic regulation of the membrane sensor VirA protein is further dissected. VirA histidine kinase and the cytoplasmic response regulator VirG protein together play a central role in regulating vir gene expression in response to phenolics. Based on a homology model of the VirA linker region, various mutant and chimeric VirA proteins were generated and examined for their ability to induce VirB promoter activity. The ability of VirA to sense and respond to three separate input signals, phenolics, sugars, and environmental pH, plays a significant role in securing successful infection. Agrobacterium attachment to plant cells is an important early step in crown gall disease progression. Motile bacteria swim toward host cells and then physically interact with host cells to form aggregates and establish a multicellular bacterial community known as a biofilm. Various genetic and environmental factors that affect Agrobacterium attachment and biofilm formation are reviewed in the article by Heindl et al. (2014). The functions of different types of exopolysaccharides that constitute the biofilm and underlying mechanisms involving how the second messenger cyclic-di-GMP, the ChvG/ChvI system, phosphorus levels, and oxygen tension influence bacterial attachment and virulence are also summarized. In the review article by Matthysse (2014), early studies and current knowledge of the mechanisms of polar and lateral bacterial attachment are summarized. These two mechanisms both contribute to bacterial attachment. When the environmental calcium and phosphate levels and pH values are low, polar attachment predominates. In addition, the phospholipids (PLs), phosphatidylcholine (PC), and phosphate-free lipid ornithine lipids (OLs) contribute to Agrobacterium virulence. In the review by Aktas et al. (2014), the biosynthetic pathways and the physiological roles of these membrane lipids are summarized. The typical eukaryotic membrane lipid PC is not frequently found in bacteria, but it constitutes almost 22% of the Agrobacterium membrane lipid. Interestingly, PCs and OLs may play opposite roles in Agrobacterium virulence. The reduction of tumor formation in a PC-deficient Agrobacterium mutant may result from impaired vir gene expressions controlled by VirA/VirG. The absence of OLs in A. tumefaciens may decrease host defense responses and therefore cause earlier and larger tumor formation. Plant cells have a variety of receptors that recognize so-called microbe- or pathogen-associated molecular patterns (MAMPs or PAMPs), and subsequently activate plant defense responses, a process known as Pattern-recognition receptor-Triggered Immunity (PTI) (Boller and Felix, 2009; Boller and He, 2009). Agrobacteriium may utilize effectors to hijack plant systems and evade plant defense responses. Pitzschke (2013) reviews strategies used by Agrobacterium to turn plant defense responses to its own advantage. Infected plant cells initiate a mitogen-activated protein kinase signaling cascade that causes VIP1 (Agrobacterium VirE2-interacting protein 1) phosphorylation and translocation into the plant nucleus to induce defense gene expression. On the other hand, Agrobacterium may hijack VIP1 to help T-DNA enter the plant nucleus. Based on the current knowledge of plant defense responses against Agrobacterium infection, Pitzschke (2013) discusses several biotechnological approaches to increase transformation efficiency. In another review by Gohlke and Deeken (2014), early plant responses to Agrobacterium, including various defense responses, hypersensitive responses, and phytohormone level alterations are discussed. The alterations in plant morphology, nutrient translocation, and metabolism caused by crown gall tumor formation are also reviewed. The authors summarize important genomic, epigenomic, transcriptomic, and metabolomic studies that reveal epigenetic changes associated with T-DNA integration and gall development. Subsequently, Hwang et al. (2015) review important pathogenic elicitors, host cell receptor molecules, and their downstream signal transduction pathways in host plants during the PAMP-triggered immune response. They highlight recent discoveries linking plant immunity to endomembrane trafficking and actin dynamic changes. Effects of both the host physiology, including hormone levels, circadian clock, developmental stages, and environmental factors, including light exposure lengths and temperature, on plant defense responses and bacterial virulence are reviewed and discussed. In nature, evidence of ancient horizontal gene transfers (HGT) from Agrobacterium to plants has been observed in the genera Nicotiana and Linaria. Sequences homologous to mikimopine-type Agrobacterium rhizogenes pRiA4 T-DNA were first discovered in the genome of untransformed tree tobacco, Nicotiana glauca, and named “cellular T-DNA” (cT-DNA; White et al., 1983). Matveeva and Lutova (2014) review cT-DNA organization, distribution, expression regulation, and a possible correlation with genetic tumor formation in Nicotiana species. They also review recent findings of cT-DNA in the genomes of Linaria species and in other dicotyledonous families. The authors suggest that plants maintaining cT-DNA in their genomes may potentially benefit microorganisms in the rhizosphere by secreting opines in the root zone. They also propose that footprints of ancient pRi T-DNA insertions in the plant genome may provide selective advantage to these plants. With this Research Topic we provide a platform for scientists to share their understanding of Agrobacterium biology and how Agrobacterium transforms plants. These contributions demonstrate how a highly active research community in plant and microbial sciences can elucidate important pathogenesis questions. Future research on Agrobacteium will continue to advance our understanding of plant-pathogen interactions, and provide new insights useful for plant genetic engineering.

Transgenic Arabidopsis plants expressing Agrobacterium tumefaciens VirD2 protein are less susceptible to Agrobacterium transformation

SUMMARY Agrobacterium tumefaciens causes crown gall disease on many plant species and can result in considerable economic losses. Here we report a new strategy to control crown gall disease by over-expressing Agrobacterium tumefaciens VirD2 protein in plants. Transgenic Arabidopsis plants over-expressing virD2 from constitutive or wound-inducible promoters are less susceptible to Agrobacterium-mediated transformation. Additionally, the transient introduction of an A. tumefaciens virD2 gene in tobacco BY-2 cells reduces subsequent Agrobacterium-mediated transformation.

Agrobacterium-Mediated Plant Transformation: Biology and Applications

Plant genetic transformation heavily relies on the bacterial pathogen Agrobacterium tumefaciens as a powerful tool to deliver genes of interest into a host plant. Inside the plant nucleus, the transferred DNA is capable of integrating into the plant genome for inheritance to the next generation (i.e. stable transformation). Alternatively, the foreign DNA can transiently remain in the nucleus without integrating into the genome but still be transcribed to produce desirable gene products (i.e. transient transformation). From the discovery of A. tumefaciens to its wide application in plant biotechnology, numerous aspects of the interaction between A. tumefaciens and plants have been elucidated. This article aims to provide a comprehensive review of the biology and the applications of Agrobacterium-mediated plant transformation, which may be useful for both microbiologists and plant biologists who desire a better understanding of plant transformation, protein expression in plants, and plant-microbe interaction.

Overexpression of the HspL Promotes Agrobacterium tumefaciens Virulence in Arabidopsis Under Heat Shock Conditions.

Agrobacterium tumefaciens transfers a specific DNA fragment from the resident tumor-inducing (Ti) plasmid and effector virulence (Vir) proteins to plant cells during infection. A. tumefaciens VirB1-11 and VirD4 proteins assemble as the type IV secretion system (T4SS), which mediates transfer of the T-DNA and effector Vir protein into plant cells, thus resulting in crown gall disease in plants. Previous studies revealed that an α-crystallin-type, small heat-shock protein (HspL) is a more effective VirB8 chaperone than three other small heat-shock proteins (HspC, HspAT1, and HspAT2). Additionally, HspL contributes to efficient T4SS-mediated DNA transfer and tumorigenesis under room-temperature growth. In this study, we aimed to characterize the impact of HspL on Agrobacterium-mediated transformation efficiency under heat-shock treatment. During heat shock, transient transformation efficiency and VirB8 protein accumulation were lower in the hspL deletion mutant than in the wild type. Overexpression of HspL in A. tumefaciens enhanced the transient transformation efficiency in root explants of both susceptible and recalcitrant Arabidopsis ecotypes. In addition, the reduced transient transformation efficiency during heat stress was recovered by overexpression of HspL in A. tumefaciens. HspL may help maintain VirB8 homeostasis and elevate Agrobacterium-mediated transformation efficiency under both heat-shock and nonheat-shock growth.

FXYD8, a Novel Regulator of Renal Na+/K+-ATPase in the Euryhaline Teleost, Tetraodon nigroviridis

FXYD proteins are important regulators of Na+/K+-ATPase (NKA) activity in mammals. As an inhabitant of estuaries, the pufferfish (Tetraodon nigroviridis) responds to ambient salinity changes with efficient osmoregulation, including alterations in branchial, and renal NKA activities. Previous studies on teleostean FXYDs have mainly focused on the expression and potential functions of FXYD proteins in gills. The goal of the present study was to elucidate the potential role of FXYD8, a member of the fish FXYD protein family, in the modulation of NKA activity in the kidneys of this euryhaline pufferfish by using molecular, biochemical, and physiological approaches. The results demonstrate that T. nigroviridis FXYD8 (TnFXYD8) interacts with NKA in renal tubules. Meanwhile, the protein expression of renal TnFXYD8 was found to be significantly upregulated in hyperosmotic seawater-acclimated pufferfish. Moreover, overexpression of TnFXYD8 in Xenopus oocytes decreased NKA activity. Our results suggest the FXYD8 is able to modulate NKA activity through inhibitory effects upon salinity challenge. The present study further extends our understanding of the functions of FXYD proteins, the regulators of NKA, in vertebrates.

Arabidopsis RETICULON-LIKE3 (RTNLB3) and RTNLB8 Participate in Agrobacterium-Mediated Plant Transformation

Agrobacterium tumefaciens can genetically transform various eukaryotic cells because of the presence of a resident tumor-inducing (Ti) plasmid. During infection, a defined region of the Ti plasmid, transfer DNA (T-DNA), is transferred from bacteria into plant cells and causes plant cells to abnormally synthesize auxin and cytokinin, which results in crown gall disease. T-DNA and several virulence (Vir) proteins are secreted through a type IV secretion system (T4SS) composed of T-pilus and a transmembrane protein complex. Three members of Arabidopsis reticulon-like B (RTNLB) proteins, RTNLB1, 2, and 4, interact with VirB2, the major component of T-pilus. Here, we have identified that other RTNLB proteins, RTNLB3 and 8, interact with VirB2 in vitro. Root-based A. tumefaciens transformation assays with Arabidopsis rtnlb3, or rtnlb5-10 single mutants showed that the rtnlb8 mutant was resistant to A. tumefaciens infection. In addition, rtnlb3 and rtnlb8 mutants showed reduced transient transformation efficiency in seedlings. RTNLB3- or 8 overexpression transgenic plants showed increased susceptibility to A. tumefaciens and Pseudomonas syringae infection. RTNLB1-4 and 8 transcript levels differed in roots, rosette leaves, cauline leaves, inflorescence, flowers, and siliques of wild-type plants. Taken together, RTNLB3 and 8 may participate in A. tumefaciens infection but may have different roles in plants.