EFB530 Plant Physiology

Agrobacterium and crown gall disease

Crown gall disease occurs when the soil bacterium Agrobacterium tumefaciens enters a stem through a wound site (crown is the stem just above the soil surface)

• causes proliferation of tissue, like cancer growth = gall
• gall can reduce the productivity of the plant
• can occur on many dicot species
• important pathogen of grape, fruit and nut trees; I have some on euonymous vines in my front yard

• infection of root tissue causes severe proliferation of roots
• continues even in absence of bacteria

What does this have to do with hormones?

• can heat a gall to 42C, kills Agrobacterium, but not plant cells, gall continues to grow and proliferate even in absence of bacteria
• gall tissue cells can divide and grow on tissue culture medium without hormones (they have no requirement for auxin or cytokinin)
• gall tissue contains abnormally high amounts of auxin and cytokinin and have the machinery to synthesize those hormones
• this machinery is gained by the stable transfer of genes from Agrobacterium to the infected plant cells

Agrobacterium has the ability to transfer DNA into the plant cells

• the DNA from the bacterium is integrated into a plant chromosome
• the transferred DNA (T-DNA) is a portion of a large plasmid
• in Agrobacterium tumefaciens, this is the Ti (=tumor-inducing) plasmid

The T-DNA includes genes for enzymes involved in IAA, cytokinin, and opine biosynthesis

• two genes, iaaM and iaaH, encode enzymes for the production of IAA
• one gene, iptZ, encodes an enzyme for the production of cytokinin
• these genes are different from the plant genes for hormone synthesis enzymes
• these genes on the bacterial Ti plasmid have eucaryotic plant-type promoters
• another gene encodes octopine synthetase, an enzyme for the production of the opine, octopine, a modified amino acid

The overproduction of hormones causes cell proliferation, while the opine produced serves as a source of nitrogen for the bacterium, but not for the plant

Mutations in individual genes of the T-DNA can cause different types of tumors to grow

• mutations in the iaa genes result in shooty tumors
• mutations in the ipt gene causes rooty tumors

• the genes for hormone and opine synthesis are removed from the Ti plasmid
• they are replaced with a selectable marker, a gene to allow for growth of only transformed cells, usually a drug- or herbicide-resistance gene (kanamycin resistance)
• can also add in another gene, from any source, as long as it has a plant promoter (such as herbicide resistance, insecticidal proteins, vitamin synthesis, etc.)
• transform Agrobacterium with this modified plasmid, then dip leaf disks in a suspension of the Agrobacterium
• the bacterium transfers the T-DNA with these genes into the plant cells and the genes become integrated into the plant chromosome
• the leaf disks are placed on tissue culture medium with the drug, so only drug-resistant cells grow
• with the appropriate addition of hormones, leaf cells can grow and eventually develop into shoots and roots, regenerating a whole plant that is transgenic

Agrobacterium-mediated transformation is only effective among plants that the bacterium can infect

• this includes most dicots

Monocots (like maize, rice, and wheat) are not susceptible to Agrobacterium and have not been effectively transformed this way

Instead, one may coat gold or tungsten particles with DNA, then "shoot" these into plant tissue = biolistic transformation

• at a low frequency, this DNA is integrated into the plant chromosome, allowing for transformation of species that have not worked with Agrobacterium

Back to EFB530 Syllabus