Identification And Characterization Of The Pathogenicity-related Genes In Lonsdalea Quercina N-5-1

Populus xeuramericana’Neva’and Populus×euramericana’zhonglin46’are important fast growing tree species in North of China. In2006, poplar bark canker was first discovered in Henan province, China, and the disease is reported for the first in the world. Lonsdalea quercina was identified as the pathogen of poplar bark canker, and pathogenic strain N-5-1isolated from spontaneous diseased poplar branches from the Henan Puyang. In this study the genome of strain N-5-1was sequenced, and22genes were suspected of the virulence factor through the analysis of genome sequence. Insertional mutagenesis of the22genes and corresponding complementation were constructed, respectively. Plant assay showed that type II secretion system (T2SS) and type III secretion system (T3SS) were essential pathogenic factor. Further studies revealed that strain N-5-1possesses complete T2SS and T3SS. To explore the relationship between strain N-5-1T2SS, T3SS and pathogenicity, these deletion mutants and complementary of structure genes gspE of T2SS, hrcV of T3SS, and genes coding protein which secreted by T2SS and T3SS were constructed, and the characteres of the effector of T3SS HrpW were studed deeply. The results are as the follows:(1) Based on the analysis of strain N-5-1genome, a complete type Ⅲ secretion system (T3SS) cluster, which is highly similar to that in pathogenic bacteria Dickeya dadantii3937and Erwinia amylovora CFBP1430, was identified in strain N-5-1. The T3SS of N-5-1is of about23kb in size including27genes,9of which are highly conservative hrc genes. Pathogenicity assay on poplar branches indicated that the△hrcV mutant was significantly less virulent than its parent, while the complemented mutant HBhrcV restored the virulence to the wild-type level. The hrcV mutation abolished the HR elicitation activity on tabacco, but the ability of growth, motility and biofilm formation was not influenced. This study suggested that the T3SS is an essential pathogenic factor in L. quercina N-5-1.(2) HrpW is a putative effector of T3SS in strain N-5-1, which has a typical harpin domain at the amino terminal and a pectate lyase (Pel) domain at its carboxyl terminal. Genetic evidence revealed that, compared to the wild-type and the complementary strain, the hrpW mutation caused a fractional but significant virulence reduction when inoculated on the poplar branches. Amino terminal domain of HrpW, but not the carboxyl terminal domain, had the ability to trigger tobacco hypersensitive response (HR). Unlike most of the HrpW homologs in other bacteria, carboxyl terminal domain of HrpW of strain N-5-1exhibited a detectable pectate lyase activity. Site-direction mutations of the104Trp and the171Trp risiduses further demonstrated that the two tryptophan residues are essential for its Pel activity. Our results suggest that HrpW in L. quercina N-5-1possesses Pel activity and is required for expression of its full virulence.(3) Strain N-5-1possesses T2SS that involved in12genes, and it has Sec-dependent and Twin argininetranslocation (Tat) two protein transport pathway. Plant assay showed that the pathogenicity of the deletion mutant gspE was decreased significantly, and the complementary strain could recover the pothogenicity. The genetic evidences proved that there is a direct relationship between the pathogenicity of strain N-5-1and T2SS.(4) Three pectate lyase genes were discovered by the analysis of strain N-5-1genome sequence, and2pel genes (pel1, pel2) of them having a high homology with the pelD, pelA, pelE of Dickeya dadantii3937, while the identity of pel3and D. zeae Echl591strain reached78%. And the proteins PelA, PelD, PelE of D. dadantii3937were secreted by T2SS. Plant assay suggested that comparing with wild strain N-5-1the pathogenicity of the pell, pel2and pel3deletion mutants has no differences. Pectate lyase activity assay at230nm wavelength showed that the pectate lyase activity of△gspE was significantly decreased, and the pectate lyase activity of△pell, Apel2, Apel3and wild strains N-5-1stayed the same level. It’s indicated that extracellular Pel proteins are secreted though T2SS, and single Pel protein in strain N-5-1has no influence on the whole pectate lyase activity and also no influence on the pothogenicity.