Agrobacterium tumefaciens

Bacteria multiply primarily in soil, not in tumors Able saprophyte Attracted to wounds Infects and transfers T-DNA from Ti plasmid to plant cells, resulting in synthesis of plant growth regulators (hormones) for undifferentiated growth (tumors), and biosynthesis of opines (nutrient source for bacteria) Bacteria multiply primarily in soil, not in tumors Very broad host range most dicots and a few monocots Ti plasmid can be manipulated to customize transferred DNA and make use of A. t. as genetic engineering tool Ti plasmid is the virulence determining factor and can be transferred from one strain to another strain, making the recipient virulent and able to infect.

Agrobacterium tumefaciens Crown gall of tomato Agrobacterium tumefaciens Rose Tobacco

Infection process (a) Chemical recognition of host and activation of virulence gene expression, (b) physical recognition and interaction between bacterium and host, (c) production of transferred substrates and transfer machinery, (d ) transfer of substrates out of the bacterium and into the host cell, (e) movement of substrates into nucleus, (f) integration of T-DNA into host genome, (g) expression of T-DNA.

Signals that induce vir gene expression Low pH Low PO4 Acetosyringone VirAG two component system receptor and kinase dimers two trans membrane domains kinase self inhibition and with the phenols phosphorylation on virG; chvE responding to sugar low pH making the phenol signaling easier. Wound conditions Necessary structural elements decide the specificity Diverse inducing signals decide the broad host range McCullen and Binns (2006) Annu. Rev. Cell Dev. Biol. 22:101-127

Type IV secretion system (T4SS) VirD2-T-strand, VirE2, VirE3, VirF, VirD5 Recruiter for D4 and the ATPase providing the energy to translocate the macromolecules Physical interaction evidences Polar location McCullen and Binns (2006) Annu. Rev. Cell Dev. Biol. 22:101-127

McCullen and Binns (2006) Annu. Rev. Cell Dev. Biol. 22:101-127 Physical attachment surface beta-1,2-glucan. Transferred macromolecules structure McCullen and Binns (2006) Annu. Rev. Cell Dev. Biol. 22:101-127

Type IV secretion is conserved Function could be diverse but the cluster structure and the protein sequences are conserved among different strains

Decreased expression or overexpression of more than 100 plant genes can alter susceptibility rat (recalcitrant to Agrobacterium transformation) genes (attachment, transfer, integration) importins PP2C (virD2) overexpression -> decreased nuclear targeting. Mutation - >higher susceptibility to Agro infection VIP1(b-zip) - (virE2)overexpression ->increased susceptibility. Piggybacks virE2 into nucleus? skp1 - (virF) cell cycle setting, targeting for proteosome? hat (hypersusceptible to Agrobacterium transformation) genes (e.g., MTF, WRKY – plant defense)

Agrobacterium exploits a defense response Dafny-Yelin et al. (2008) Trends Plant Sci. 13:102-105

Dimerization of VirD2 Binding Protein Is Essential for Agrobacterium Induced Tumor Formation in Plants Padavannil et al. (2014) PLoS Pathog 10: e1003948.

A. tumefaciens responses to plant-derived signaling molecules Phenolic compound can increase the Ti plasmid copies. Opine can activate the opine metabolism genes, as well as quorum sensing system to induce the Ti conjugation. QS also increase the Ti copy numbers. SA GABA activate the quorum quenching, downregulate the QS; SA IAA EA downregulate Vir gene Subramoni et al (2014) Front Plant Sci. 5: 322.

Remaining questions Mechanism of T-DNA transfer and integration A. t. chromosomal genes involved in pathogenesis and host range determination (transformation efficiency) Plant genes involved in all aspects, especially: Host specificity Integration (primary limiting factor) (Can alteration of req’d plant genes confer resistance to crown gall?) (Can alteration of req’d plant genes improve transformation?) Coevolution