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A potyvirus (isolate IB) causing dappled or faded fruits and foliar mosaic symptoms of purple passionfruit, was found in the botanical garden of Kagoshima University, Japan. This isolate--differed in host range from isolates of Passion fruit woodiness virus (PWV)-AO, previously reported to cause "woodiness" in Japan. Isolates IB and AO had 83% amino acid identity in their coat proteins (CPs). In phylogenetic analysis, East Asian isolates IB, AO, and PWV-Taiwan clustered together, and were distinguishable from Australian PWV and Brazilian Cowpea aphid-borne mosaic virus isolates, which also cause "woodiness" in passionfruit. We propose the name "East Asian Passiflora virus (EAPV)" for the new potyvirus species.

In this study, we clarified the functions of three uncharacterized enzymes, XCV2724, XCV2728, and XCV2729, in Xanthomonas euvesicatoria, the causal agent of bacterial spot of tomato and pepper. The genes corresponding to the three enzymes are homologs of hypBA1, hypBA2, and hypAA from Bifidobacterium longum and are unique to Xanthomonas spp. among plant pathogenic bacteria. Functional characterization of the recombinant enzymes expressed using microbial systems revealed that they degrade the arabinofurano-oligosaccharides present on hydroxyproline (Hyp)-rich glycoproteins (HRGPs) such as extensin and solanaceous lectins in plant cell walls. These enzymes work coordinately to degrade the oligosaccharides. First, XeHypAA (XCV2728), belonging to the glycoside hydrolase (GH) 43 family, releases L-arabinose from L-arabinofuranose (Araf)-α1,3-Araf-ß1,2-Araf-ß1,2-Araf-ß-Hyp (Ara4-Hyp), cleaving its α1,3 bond; second, XeHypBA2 (XCV2729), belonging to the GH121 family, releases the disaccharide Araf-ß1,2-Araf from Araf-ß1,2-Araf-ß1,2-Araf-ß-Hyp (Ara3-Hyp); finally, XeHypBA1 (XCV2724), belonging to GH family 127, releases L-arabinose from Araf-ß-Hyp (Ara-Hyp). In summary, the main oligosaccharide structure of Ara4-Hyp on the HRGPs is degraded to Ara3-Hyp, then to Ara-Hyp, and finally to Ara monosaccharides by the action of these three enzymes. HRGPs containing oligosaccharide substrates have been reported to contribute to plant defense, and interestingly, the promoter region of the operon (xehypBA2 and xehypAA) contains the plant-inducible promoter box for binding the regulator protein HrpX involved in pathogenicity. We then analyzed the expression level of the operon gene in hrp-inducing medium and in plants and constructed gene-deletion mutants. However, although the operon was evidently upregulated by HrpX, three single-gene deletion mutants (ΔxehypBA1, ΔxehypBA2, ΔxehypAA) and even a triple-gene deletion mutant (ΔxehypBA1-BA2-AA) remained pathogenic, and had no effect on nonhost resistance, either, indicating that these three enzymes are not involved in either pathogenicity or nonhost resistance reactions. This is the first report of enzymes in plant pathogenic bacteria that catalyze the degradation of Hyp-linked-L-arabinofuranosides in plant cell walls.

The Ibusuki (IB) strain of the East Asian Passiflora virus (EAPV) causes mottling of fruit when it infects passionfruit, but not malformation or woodiness, unlike the Amami-O-shima (AO) strain, and the host range for these two strains are different. We determined the complete nucleotide sequence of the IB strain, and a comparison with that of the AO strain revealed the great diversity of the 5'-terminal region of the IB strain's genome (5' UTR and P1 gene). The involvement of these regions in the different symptoms on fruit and host range was suggested. The neighbor-joining tree constructed using the nucleotide sequences of coat protein gene of eight EAPV isolates including those from abroad showed the independent position of the IB strain, and that constructed using the whole ORFs also showed distant relationships between the AO and IB strains. We investigated the distribution of the two strains in southern Japan from 2005 to 2010. The AO strain was detected in the samples from AO at all periods, and its emergence was also observed in the Kagoshima mainland in 2005. In contrast, the IB strain is restricted to the Kagoshima mainland, and the distribution survey revealed that this strain is now extinct even in this region, indicating the uniqueness of the IB strain in terms of sequence properties and geographical distribution.

summary Geotrichum candidum citrus race, a fungus that causes a sour rot disease in citrus fruits, secretes an endo-polygalacturonase (PG) that may facilitate the disease. There also exists a non-citrus race that is non-pathogenic to citrus fruits. In this research, we found that the PG activity of the citrus race isolates was much higher than that of the non-citrus race isolates in culture medium and inoculated lemon peel, and that there was a significant correlation between the PG activity and pathogenicity. We isolated the two corresponding PG genes, S31pg1 and S63pg1, from citrus race S31 and non-citrus race S63, respectively. S31PG1 and S63PG1 consisted of 368 and 369 amino acids, respectively. The two PG genes showed 68% identity at the amino acid level. In expression studies, S31pg1 transcript was detected in mycelia grown in liquid cultures of citrus race S31 containing either glucose, pectin or lemon peel broth. The transcript was also detected in lemon peel inoculated with the isolate. On the other hand, no transcript of S63pg1 was detected in mycelia grown on any liquid cultures of non-citrus race S63 and lemon peel inoculated with the isolate. These results indicate that PG may play an important role in the development of the sour rot symptom and be involved in the difference of pathogenicity between the two races.

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The CRISPR-Cas9 system has a potential for wide application in organisms that particularly present low homologous integration rates. In this study, we developed three different methods using this system to replace a gene through homology-directed repair in the plant pathogenic fungus Colletotrichum sansevieriae, which has a low recombination frequency. The gene encoding scytalone dehydratase was used as the target so that mutants can be readily distinguished owning to a lack of melanin biosynthesis. First, we performed a plasmid-based method using plasmids containing a Cas9 expression cassette and/or a single-guide RNA (sgRNA) under the control of the endogenous U6 snRNA promoter, and 67 out of 69 (97.1%) transformants exhibited a melanin-deficient phenotype with high efficiency. Second, we performed a transformation using a Cas9 protein/sgRNA complex and obtained 23 out of 28 (82.1%) transformants. Lastly, we developed a hybrid system combining a Cas9 protein and donor DNA-sgRNA expression plasmid, which yielded 75 out of 84 (89.2%) transformants. This system was also applicable to four other genes at different loci of the fungus. This is the first study to establish a CRISPR/Cas9 gene replacement system in Colletotrichum spp. and it presents a potential application for a broad range of use in other species of the genus.

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