Functional Analysis Of A Novel Gene Cluster Involved In LPS Biosynthesis Of Xanthomonas Oryzae Pv. Oryzicola Strain RS105

Xanthomonas oryzae comprises two pathovars, X. o. pv. oryzae (Xoo) and X. o. pv. oryzicola (Xooc). They cause leaf blight and leaf streak symptoms on rice respectively. Rice bacterial leaf blight is an important disease in subtropical areas including China. It may cause over 50% production loss. Bacterial leaf streak is also an important disease on rice in the tropical areas, including Bengal, Cambodia, south of China such as Fujian, Guangdong and Hainan. Losses of 5-30% have been reported when rice was infected with Xooc. Xoo and Xooc are important models of phytopathogen in studying bacteria-host interaction. To understand the mechanism of Xoo and Xooc is crucial for developing strategies to prevent plant disease. However, the restriction -modification system that exists in Xoo and Xooc rejects foreign DNA transformation into their genomic DNA. This feature holds back us to learn the mechanism of pathogenicity on the basis of molecular level by use of reverse genetic manipulation. In this paper, reverse genetic experiments were used to study the role of LPS biosynthersis in bacterial virulence. These results will be part of the groundwork to understand Xooc and Xoo.1 Screening of exopolysaccharide and virulence deficient mutants and positive clonesNine mutants deficient in EPS biosynthesis and pathogenicity on rice (EPSˉPathˉ) were obtained with ethyl methane sulfonate (EMS) mutagenesis. Among which, 6 mutants (M12, M13, M17, M19, M21, M23) were screened from Xooc strain RS105 and 3 (K7, K21, K37) from Xoo strain PXO99. By genomic DNA library complementation with the selected mutant strains, a positive clone P35 was obtained from 2200 library clones of RS105 to complement the deficient mutant M12, and a positive clone A286 was selected from 2000 clones of PXO99 genomic library to complement the mutant K.7.Subclones of P35 completely digested by either KpnI or EcoRI could not restore the deficient functions of M12. A 12.7kb KpnI partially digested clone PK23, which covered two KpnI fragments restored the deficient functions of M12. Further sub-cloning of PK23...