Fire blight, caused by bacterium Erwinia amylovora, is a very serious disease affecting apple, pear and other fruit plants. The development of phage-based biopesticides is currently in progress in our lab. Emergence of phage-resistant bacteria is a valid concern. Two attributes of the bacterial host that may contribute to the development of resistance were studied, the Clustered Regularly Interspaced Short Palindromic Repeats/ CRISPR-associated (CRISPR/Cas) system and exopolysaccharide (EPS) interaction with phages.
The structure of E. amylovora CRISPR/Cas system was determined in 8 E. amylovora isolates from different geographical regions. Three CRISPR-array sets named CR1, CR2 and CR3 were detected in 4 isolates, and only 2 arrays were determined in the rest of the isolates. No significant similarity was found between spacers in any of these systems to phage DNA sequenced in this study or from GenBank. Also the Cas level of expression was not stimulated during phage infection. Introduction of extra copies of Cas genes to enhance expression did not result in phage resistance. Nevertheless, E. amylovora CRISPR/Cas system was found to be efficient in blocking the transformation of plasmids carrying protospacers matched spacers in CRR1 and CRR2.
Among phages that have been sequenced in this study are ΦEa9-2 and ΦEa35-70. ΦEa9-2 (Podoviridae) genome is 75,568 bp, and found to be related to coliphage N4. ΦEa35-70 (Myoviridae) genome is 271,084 bp, and found to carry a potential EPS depolymerase gene. Activity of ΦEa35-70 EPS depolymerase was only detected when cloned and expressed in E. coli, but His-tagged purified protein did not exhibit any EPS-depolymerase activities.
This study offers critical information for the design of novel and effective phage-based biopesticides for the control of E. amylovora. It provides a new knowledge on the molecular structure and function of CRISPR/Cas system and EPS-phage interaction
