Arabidopsis RAB8A, RAB8B and RAB8D Proteins Interact with Several RTNLB Proteins and are Involved in the Agrobacterium tumefaciens Infection Process.

Arabidopsis thaliana small GTP-binding proteins, AtRAB8s, associate with the endomembrane system and modulate tubulovesicular trafficking between compartments of the biosynthetic and endocytic pathways. There are five members in Arabidopsis, namely AtRAB8A-8E. Yeast two-hybrid assays, bimolecular fluorescence complementation assays and glutathione-S-transferase pull-down assays showed that RAB8A, 8B and 8D interacted with several membrane-associated reticulon-like (AtRTNLB) proteins in yeast, plant cells and in vitro. Furthermore, RAB8A, 8B and 8D proteins showed interactions with the Agrobacterium tumefaciens virulence protein, VirB2, a component of a type IV secretion system (T4SS). A. tumefaciens uses a T4SS to transfer T-DNA and Virulence proteins to plants, which causes crown gall disease in plants. The Arabidopsis rab8A, rab8B and rab8D single mutants showed decreased levels of Agrobacterium-mediated root and seedling transformation, while the RAB8A, 8B and 8D overexpression transgenic Arabidopsis plants were hypersusceptible to A. tumefaciens and Pseudomonas syringae infections. RAB8A-8E transcripts accumulated differently in roots, rosette leaves, cauline leaves, inflorescence and flowers of wild-type plants. In summary, RAB8A, 8B and 8D interacted with several RTNLB proteins and participated in A. tumefaciens and P. syringae infection processes.

Functional analysis of McSnRK1 (SNF1-related protein kinase 1) in regulating Na/K homeostasis in transgenic cultured cells and roots of halophyte Mesembryanthemum crystallinum.

KEY MESSAGE: Transgenic callus and roots of ice plant with altered SnRK1 function were established using Agrobacterium-mediated transformation. The role of McSnRK1 in controlling Na+ influx and Na/K ratio was demonstrated. SnRK1 kinases (SNF1-related protein kinase1) control metabolic adaptation during energy deprivation and regulate protective mechanisms against environmental stress. Yeast SNF1 activates a P-type ATPase, the Na+ exclusion pump, under glucose starvation. The involvement of plant SnRK1 in salt stress response is largely unknown. We previously identified a salt-induced McSnRK1 in the halophyte ice plant (Mesembryanthemum crystallinum). In the current study, the function of McSnRK1 in salt tolerance was analyzed in transgenic cultured cells and roots of ice plant. Ice plant callus constitutively expressed a high level of McSnRK1 and introducing the full-length McSnRK1 did not alter the Na/K ratio at 24 h after 200 mM NaCl treatment. However, interfering with McSnRK1 activity by introducing a truncate McSnRK1 to produce a dominant-negative form of McSnRK1 increased cellular Na+ accumulation and Na/K ratio. As a result, the growth of cultured cells diminished under salt treatment. Hydroponically grown ice plants with roots expressing full-length McSnRK1 had better growth and lowered Na/K ratio compared to the wild-type or vector-only plants. Roots expressing a truncate McSnRK1 had reduced growth and high Na/K ratio under 400 mM NaCl treatment. The changes in Na/K ratio in transgenic cells and whole plants demonstrated the function of SnRK1 in controlling Na+ flux and maintaining Na/K homeostasis under salinity. The Agrobacterium-mediated transformation system could be a versatile tool for functional analysis of genes involved in salt tolerance in the ice plant.

Correction: Huang, F.-C., et al. Arabidopsis RETICULON-LIKE3 (RTNLB3) and RTNLB8 Participate in Agrobacterium-Mediated Plant Transformation. Int. J. Mol. Sci. 2018, 19, 638.

The authors would like to make a correction to their published paper [...].

Effective Agrobacterium-mediated transformation protocols for callus and roots of halophyte ice plant (Mesembryanthemum crystallinum).

BACKGROUND: Ice plant (Mesembryanthemum crystallinum L.) is a model plant for studying salt-tolerant mechanisms in higher plants. Many salt stress-responsive ice plant genes have been identified with molecular and biochemical approaches. However, no further functional characterization of these genes in host plant due to lack of easy and effective transformation protocols. RESULTS: To establish efficient transformation system of ice plants, three types of ice plant materials, hypocotyl-derived callus, aseptically-grown seedlings and pot-grown juvenile plants, were used to develop Agrobacterium-mediated transformation protocols. The highest transient transformation efficiency was with 5-day-old ice plant callus co-incubated with an Agrobacterium tumefaciens at 2.5 × 109 cells mL-1 for 48 h. The 3-day-old ice plant seedlings with root tip removed were successfully infected with A. tumefaciens or A. rhizogenes, and obtained 85% and 33-100% transient transformation rates, respectively. The transient transformation assays in ice plant callus and seedlings demonstrated that the concentrations of Agrobacteria, the durations of co-incubation time, and the plant growth stages were three important factors affecting the transient transformation efficiencies. Additionally, pot-grown juvenile plants were syringe-injected with two A. rhizogenes strains A8196 and NCPPB 1855, to establish transformed roots. After infections, ice plants were grown hydroponically and showed GUS expressions in transformed roots for 8 consecutive weeks. CONCLUSIONS: Our Agrobacterium-mediated transformation protocols utilized hypocotyl-derived callus and seedlings as plant materials, which can be easily obtained in large quantity. The average successful transient transformation rates were about 2.4-3.0% with callus and 33.3-100.0% with seedlings. We also developed a rapid and efficient protocol to generate transgenic roots by A. rhizogenes infections without laborious and challenging tissue culture techniques. This protocol to establish composite ice plant system demonstrates excellent improvements in efficiency, efficacy, and ease of use over previous ice plant transformation protocols. These Agrobacterium-mediated transformation protocols can be versatile and efficient tools for exploring gene functions at cellular and organ levels of ice plants.

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.