Plasmid-based and -free methods using CRISPR/Cas9 system for replacement of targeted genes in Colletotrichum sansevieriae.

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.

Functional characterization of unique enzymes in Xanthomonas euvesicatoria related to degradation of arabinofurano-oligosaccharides on hydroxyproline-rich glycoproteins.

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.

Draft genome sequence of Colletotrichum sansevieriae Sa-1-2, the anthracnose pathogen of Sansevieria trifasciata.

Colletotrichum sansevieriae is an ascomycete fungus causing anthracnose disease on plants in the genus Sansevieria. Here, we report the draft genome sequence of isolate Sa-1-2 of this fungus. The genome size is >51 Mb, and the assembly consists of 8647 contigs and contains 13,664 predicted protein-coding genes. Pathogenicity factors such as plant cell wall-degrading enzymes and effector proteins were also predicted. Additionally, the phylogenetic relationship of isolates from different Colletotrichum spp. was analyzed, revealing that the isolate belongs to a novel major clade consisting of species that infect succulent plants originating from Africa. The draft genome sequence has been deposited at GenBank under accession number NJHP00000000.

Detection of Cucurbit chlorotic yellows virus from Bemisia tabaci captured on sticky traps using reverse transcription loop-mediated isothermal amplification (RT-LAMP) and simple template preparation.

Cucurbit chlorotic yellows virus (CCYV) of the genus Crinivirus within the family Closteroviridae is an emerging infectious agent of cucurbits leading to severe disease and significant economic losses. Effective detection and identification methods for this virus are urgently required. In this study, a reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay was developed to detect CCYV from its vector Bemisia tabaci. LAMP primer sets to detect CCYV were evaluated for their sensitivity and specificity, and a primer set designed from the HSP70h gene with corresponding loop primers were selected. The RT-LAMP assay was applied to detect CCYV from viruliferous B. tabaci trapped on sticky traps. A simple extraction procedure using RNAsecure™ was developed for template preparation. CCYV was detected in all of the B. tabaci 0, 1, 7 and 14 days after they were trapped. Although the rise of turbidity was delayed in reactions using RNA from B. tabaci trapped for 7 and 14 days compared with those from 0 and 1 day, the DNA amplification was sufficient to detect CCYV in all of the samples. These findings therefore present a simple template preparation method and an effective RT-LAMP assay, which can be easily and rapidly performed to monitor CCYV-viruliferous B. tabaci in the field.

Complete nucleotide sequence of a new isolate of passion fruit woodiness virus from Western Australia.

We determined the complete genome sequence of the passion fruit woodiness virus Gld-1 isolate (PWV-Gld-1) from Australia and compared it with that of PWV-MU-2, another Australian isolate of PWV. The genomes shared high sequence identity in both the complete nucleotide sequence and the ORF amino acid sequence. All of the cleavage sites of each protein were identical to those of MU-2, and the sequence identity for the individual proteins ranged from 97.2 % to 100.0 %. However, the 5' untranslated region (5'UTR) of the Gld-1 isolate shared only 46.8 % sequence identity with that of PWV-MU-2 and was 177 nucleotides shorter. Re-sequencing of the 5'UTR of MU-2 revealed that the 5' end of the original sequence includes an artifact generated by deep sequencing.

Agrobacterium tumefaciens-mediated transformation for investigating pathogenicity genes of the phytopathogenic fungus Colletotrichum sansevieriae.

Agrobacterium tumefaciens-mediated transformation (AtMT) has become a common technique for DNA transformation of yeast and filamentous fungi. In this study, we first established a protocol of AtMT for the phytopathogenic fungus Colletotrichum sansevieriae. Binary T-DNA vector containing the hygromycin B phosphotransferase gene controlled by the Aspergillus nidulans gpdA promoter and the trpC terminator was constructed with pCAMBIA0380 and used with three different strains LBA4404, GV3101, and GV2260 of A. tumefaciens. Transformants were most effectively obtained when GV2260 and C. sansevieriae Sa-1-2 were co-cultivated; there were about 320 transformants per 10(6) spores. When 1,048 transformants were inoculated on Sansevieria trifasciata, three transformants were found to have completely lost their pathogenicity and two transformants displayed reduced pathogenicity. All of the five transformants had a single copy of T-DNA in their genomes. The three pathogenicity-deficient transformants were subjected to thermal asymmetric interlaced polymerase chain reaction and the reaction allowed us to amplify the sequences flanking the left and/or right borders. The flanking sequences of the two transformants, M154 and M875, showed no homology to any sequences in databases, but the sequences of M678 contained motifs of alpha-1,3-glucan synthase, suggesting that the gene might contribute to the pathogenicity of C. sansevieriae. This study describes a useful method for investigating pathogenicity genes in C. sansevieriae.

Molecular characterization and specific detection of two genetically distinguishable strains of East Asian Passiflora virus (EAPV) and their distribution in southern Japan.

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.