The Use of Symbionts Expressing Pear PGIP to Delay or Possibly Prevent Development of Pierce's Disease
- Author(s): Charoenkajonchai, Surachet
- Advisor(s): Miller, Thomas A
- et al.
Polygalacturonase (PG) is produced by endophytic bacteria to degrade the pit membranes of xylem vessels to facilitate movement between xylem channels. Pear polygalacturonase-inhibiting protein (pPGIP) is produced by the pear plant to block the movement of pathogenic bacteria in the plant. pPGIP is an agent being examined to protect against the pathogen X. fastidiosa from causing PD in grapevine. In the present study, candidate symbiotic bacteria were genetically altered to express pPGIP to inhibit the movement of X. fastidiosa and prevent development of PD symptoms in grapevines. Here I report successful transformation of P. agglomerans and X. fastidiosa with pPGIP plasmids, pMCS5-PGIP and pXF20-PGIP respectively. Stability of plasmid pMCS5-PGIP and pXF20-PGIP was verified by PCR analysis and gel electrophoresis. Expression of pPGIP by both of these bacteria was verified by Western blot analysis. However P. agglomerans pMCS5-PGIP did not secrete pPGIP, but Xf did secrete a very small amount of the protein. Moreover, pPGIP extracted from cell lysate and obtained from supernatant of P. agglomerans pMCS5-PGIP did not show inhibition of PG in a bioassay. Whereas, pPGIP extracted from cell lysate and obtained from supernatant of X. fastidiosa pXF20-PGIP did show inhibition of PG in a bioassay. This shows that in principle the delivery bacteria can synthesize and secrete an agent that could be use to prevent PD.
The growth of X. fastidiosa pXF20-PGIP in planta is significantly lower than X. fastidiosa. The presence of PGIP was detected in total plant extracts and in the xylem fluid of the plants inoculated with X. fastidiosa pXF20-PGIP using Western blot analysis. X. fastidiosa and X. fastidiosa pXF20-PGIP were inoculated in to grapevines. Ten weeks after inoculation, X. fastidiosa pXF20-PGIP at 45 cm above inoculation point (IP) (8.96 ±1.04253 pg/µl) were found significantly less than X. fastidiosa (54.60 ±12.01012 pg/µl) at the same distance. Bacteria in double inoculation plants, which were inoculated with X. fastidiosa pXF20-PGIP at day 0 and again with X. fastidiosa at week 4 at 45 cm above the first IP, were found significantly lower than bacteria in plants with only X. fastidiosa inoculation. The reduction of bacteria in plants with double inoculation may be due to the present of PGIP. Over time, the migration of X. fastidiosa was found in further distance in the double inoculation plants than the plant with only X. fastidiosa inoculation. In conclusion this X. fastidiosa pXF20-PGIP may contribute to inhibition of the growth and movement of X. fastidiosa, and possible delay development of Pierce's Disease.