Abstract
The kiwifruit bacterial canker pathogen, Pseudomonas syringae pv. actinidiae (Psa), causes enormous economic damages in many kiwifruit producing countries. In 2015, biovar 6, the novel biovar of Psa, was found in Nagano Prefecture, Japan. The genomes of two representative strains of biovar 6 (MAFF 212134 and MAFF 212141) were sequenced and analysed, indicating that their genomes are the most similar to that of biovar 3 among the known Psa biovars, based on average nucleotide identity analysis. Biovar 3 has neither the phaseolotoxin synthesis gene cluster nor the coronatine synthesis gene cluster, whereas biovar 6 has both clusters and produces both phytotoxins. We found that biovar 6 possesses 29 type III secreted effector (T3SE) genes, among which avrRps4 and hopBI1 are unique to biovar 6. The expression of T3SE genes and two phytotoxin synthesis gene clusters of biovar 6 during the early stages of host infection was investigated using RNA-Seq analysis, showing that these genes could be grouped into three categories: constantly expressed genes, constantly suppressed genes, and temporarily induced genes. A PCR assay was established to differentiate biovar 6 strains from the other Psa biovars and the closely related pathovar, pv. actinidifoliorum, by using avrRps4 as a biovar 6-specific marker gene.
Highlights
Kiwifruit, a type of deciduous fruit tree, belongs to the genus Actinidia and is cultivated commercially in many parts of the world, and its economic importance has been increasing in recent years
The sequences of acnB, cts, gapA, gyrB, pfk, pgi, and rpoD, which are well-known as housekeeping genes and have been used as molecular markers for phylogenetic analyses, were extracted from the genome sequences of MAFF 212134 and MAFF 212141 determined in this study and compared with those of the respective strains reported in our previous study[10], showing that they are completely identical
Thereafter, a dendrogram based on average nucleotide identity (ANI) values was constructed by using the unweighted pair group method with arithmetic mean (UPGMA); the results showed that the two strains of biovar 6 clustered independently of the other Psa biovars and Pfm, and that biovar 3 was the sister group to biovar 6 (Fig. 1)
Summary
A type of deciduous fruit tree, belongs to the genus Actinidia and is cultivated commercially in many parts of the world, and its economic importance has been increasing in recent years. Biovar 1, including the Japanese isolates that were described as Psa for the first time in the world[7], can produce phaseolotoxin, a type of phytotoxin[8]. Biovar 3 is a severe virulent group, causing pandemics throughout the world It has not been clarified so far that biovar 3 produces any known toxins[9]. Studying the genetic characteristics of biovar 6 is important for understanding the evolutionary paths and pathogenic mechanisms of phytotoxin-producing www.nature.com/scientificreports/. We sequenced the genomes of biovar 6, and compared them with those of the other Psa biovars and Pfm, especially focusing on the loci responsible for the synthesis of phytotoxins and type III secreted effectors (T3SEs), which are considered as major factors involved in pathogenicity/virulence. We investigated gene expression of biovar 6 upon its host infection and established a PCR assay to identify biovar 6
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