Agrobacterium and Rhizobium
A pathogen and a symbiont
– Influence plant cell growth, division, and differentiation
– Have value in agriculture
Agrobacterium tumefaciens
• 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
Rose
Tobacco
Crown gall of tomato
Agrobacterium tumefaciens
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
Acetosyringone
Low pH
Low PO4
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
McCullen and Binns (2006) Annu. Rev. Cell Dev. Biol. 22:101-127
McCullen and Binns (2006) Annu. Rev. Cell Dev. Biol. 22:101-127
Type IV secretion is conserved
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
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
Rhizobium spp.
• Diverse group of plant symbiotic species
• Broad range of host plants, but mostly legumes (with
exceptions - Parasponia)
• Induce formation of nodules: bulbous organs on roots
or “bumps on beans”
• Reside as “bacteroids” (irregularly shaped cells)
within nodule and fix atmospheric nitrogen into a form
useable by the plant
Symbiotic Nitrogen Fixation
A
C
B
Leguminous
plants
α-proteobacteria
Agrobacterium
Q1: Why legumes?
Q2: How specificity?
Soybean
Bean
Soybean
Determinate
Alfalfa
Indeterminate
Trivia quiz: Why are nodules pink?
Nodules
Clover
Alfalfa
Rhizosphere and infection
Attract and attach
Flavonoids from plants trigger Nod factors from rhizobia,
which in turn initiate host plant developmental changes
1) alter root hair growth (curling)
2) induce plant nod gene expression
3) stimulate plant cortical cell division
Signal Exchange
Plant flavonoids- nod gene inducer
Binding
NodD
Transcriptional
activation
nodABC
Translation
Production
NodABC
luteolin
alfalfa
genistein
soybean
Synthesis
Noe
Nol
A
lipochitinoligosaccharide
IT term: Handshaking
Signal Transduction
Jones KM et al. (2007) Nat. Rev. Microbio. 5:619
Initiate the infection thread
shepherd’s crook
rhicadhesin protein
lectin
Initiate the nodule primodia
Oldroyd et al. (2007) Science, 315:52
Nodule organogenesis
IT Elongation
Exopolysaccharides
B
Succinoglycan
Galactoglucan
K-antigen
B
A
B
Type 3 Secretion System
Host plant immune response
80000 on 10 seedlings; hundreds attached; 52 infection threads; 27 nodules
Nitrogen fixation
Oldroyd et al. (2011) Annu. Rev. Genet. 45:119-144
C
C
B
A
A
Nodule types and development
Medicago truncatula
Lotus japonicus
Popp and Ott (2011) Curr. Opin. Plant Biol. 14:458–467
snf1-1 (spontaneous nodule formation)
DMI3 (CCaMK). T->I disrupts autoinhibition
Gleason C et al (2006) Nature 441:1149-52.
Tirichine L et al (2006) Nature 441:1153-6.
HYPERINFECTED 1 (HIT1)
snf2
Murray JD et al (2007) Science 315:101-104.
Tirichine L et al (2007) Science 315:104-107.
Important questions remaining
• Bacterial
– Diversity of nod factors and host specificity
– Regulation of nod and nif genes; other genes involved in
nodulation
• Plant
– Fate of nod factors in plants
– Genes and processes involved in nodulation
• Perception
• Signal transduction
• Morphogenesis
• Autoregulation/suppression (number of nodules)
– Physiological changes - how brought about and why
important?
• Host defense suppression and subversion
– PAMPS and effectors
Evolution
How about rhizobia and legumes?