Articles
THE HRP GENE CLUSTER OF ERWINIA AMYLOVORA CAUSES ELECTROLYTE LEAKAGE AND NECROSIS IN APPLE LEAVES AND FACILITATES BACTERIAL MULTIPLICATION
Ooze production occurs within these organs, which is an indication of great increases in the pathogen population (van der Zwet and Beer, 1995). An increase in the permeability of affected plant membranes begins very early during pathogenesis and is characteristic of many plant diseases.
The increase of cell permeability, which can be assessed by measuring the conductivity of fluids surrounding plant cells, has been suggested as the mechanism of releasing nutrients to support multiplication of bacterial pathogens (Atkinson and Baker, 1987).
The entire hrp gene cluster of E. amylovora was cloned in a cosmid named pCPP430. The insert of about 40 kb of DNA seems to harbor all the genes necessary for the hrp phenotype.
When the clone is expressed in other bacteria such as Escherichia coli and Erwinia herbicola, these bacteria can cause the hypersensitive reaction (HR) in tobacco, bean, and many other plants (Beer et al., 1990). The protein harpin is synthesized and exported by the hrp gene cluster of E. amylovora. Harpin itself induces the HR in tobacco leaves and increases electrolyte leakage from tobacco cells soon after mixing with cell suspensions (Wei et al., 1992).
We studied the effect of different hrp gene clones on vacuum-infiltrated apple leaf segments.
Segment appearance, electrolyte leakage from segments and bacterial multiplication in segments were studied in response to the clones.
Four strains were used. 1. E. coli DH5 (pCPP430) contains the entire 40 kb hrp gene cluster of E. amylovora; 2. E. coli DH5(pCPP430hrpN-), the same as 1, except that hrpN, the gene encoding harpin, was mutagenized by a nonpolar insertion; 3. E. coli DH5(pCPP450), cosmid clone containing the hrp essential region but lacking the left-most 13 kb of DNA relative to pCPP430; 4. E. coli DH5(pCPP9) contains the cosmid vector for all of the above clones.
A bacterial suspension containing 108 cfu/ml of E. coli DH5(pCPP430) induced browning 16 to 24 hrs after infiltration into apple leaf segments.
After 36 to 48 hrs, complete browning and necrosis was evident and ooze-like drops had formed on the segments.
Leaf segments infiltrated with the other three strains remained green and no ooze developed as with the buffer control.
When the inoculum concentration was increased to 109 cfu/ml of E. coli DH5(pCPP430), necrosis and ooze developed faster and were more complete and profound, respectively.
Some of the segments infiltrated with the mutant (strain 2) at the higher concentration showed necrosis after 48 hrs.
Leaf segments infiltrated with strains 3 and 4 at 109 cfu/ml showed no macroscopic response.
The population E. coli DH5(pCPP430) recovered from apple leaf segments four days after infiltration at 108 cfu/ml was at least one order of magnitude greater than the