First report of pepper yellow leaf curl Thailand virus infecting hot chili in central Vietnam and Laos.

Hot chili pepper (Capsicum annuum) cultivation has been on the rise in South East Asia to meet export demands. In Thailand, the top chili exporter in South East Asia, chili production has been severely hampered by pepper yellow leaf curl disease (YLCD) caused by the begomovirus pepper yellow leaf curl Thailand virus (PepYLCThV) (Chiemsombat et al., 2018; Suwor et al., 2021). In the neighbouring countries of Laos and Vietnam, a limited survey of chili fields (200 plants in total) in Savannakhet (Savannakhet University campus, n = 150), Laos and Quang Nam province (Ka Dang commune, Dong Giang district, n = 50), central Vietnam in 2023 led to the finding of eight plants (5 in Laos and 3 in Vietnam) exhibiting YLCD-like symptoms, which included bright yellow color in young leaves and leaf curl and mosaic chlorosis in mature leaves (Fig. S1). Total DNA was extracted from leaves of two symptomatic plants (one from Savannakhet and one from Quang Nam) using a cetyltrimethylammonium bromide-based DNA extraction protocol (Doyle & Doyle, 1987; Nguyen et al., 2023). Next, PCR were performed using newly designed PepYLCThV-specific primers based on PepYLCThV sequences in GenBank (Table 1). PCR products of expected sizes were observed in samples with disease symptoms, but not from DNA extracted from C. annuum (cv. VA.99999) grown at the Institute of Biotechnology in Thua Thien Hue, Vietnam (Fig. S2). The amplicons were Sanger sequenced (Apical Scientific, Selangor, Malaysia) and the complete bipartite genome sequence of two isolates ('Sava01' from Laos and 'QNam01' from Vietnam) were obtained. The sequences of the DNA-A component from isolates 'Sava01' (GenBank PP437580) and 'QNam01' (GenBank PP437581) exhibited the highest sequence identity of 99.2% and 94.7% with the PepYLCThV isolate 'ChiangDaoS1' (GenBank OM677627), respectively (Table 2). Conversely, the sequences of the DNA-B component from the isolates 'Sava01' (GenBank PP437579) and 'QNam01' (GenBank PP437582) exhibited the highest similarity of 91.8% and 90.9% with the PepYLCThV isolate 'KKN601' (GenBank MW715820), respectively (Table 2). These results confirmed the presence of PepYLCThV in hot chili pepper plants exhibiting YLCD-like symptoms in central Vietnam and Laos. Infectious clones of PepYLCThV DNA-A and DNA-B (isolate 'QNam01') were created based on the pLX-AS vector as described by Pasin (2022), and transformed into Agrobacterium tumefaciens EHA105. The resulting bacteria were cultured in LB broth containing rifampicin (25 µg/mL) and kanamycin (50 µg/mL) at 28°C and used for agroinoculation of Nicotiana benthamiana (n = 6) and C. annuum (cv. VA.99999, n = 6) (4-6 leaf plants) as described by Pasin (2022). In all N. benthamiana plants, agroinoculation with both DNA-A and DNA-B infectious clones caused stunted growth, severe leaf curl, with yellow and white patches 21 days post inoculation (Fig. S3). In C. annuum plants, symptom expression, which included leaf curl and stunted leaves with yellow mosaic patterns, was observed in two out of six inoculated plants six weeks postinoculation (Fig. S3). PCR assays confirmed the presence of PepYLCThV DNA in N. benthamiana and C. annuum symptomatic leaves (Fig. S4). To our knowledge, this is the first report of pepper yellow leaf curl Thailand virus in hot chili pepper in Laos and central Vietnam. Appropriate containment and management strategies should be developed and implemented to control the spread of this disease in hot chili pepper crops in both countries.

Morphological, phytochemical and genetic characterization of Centella asiatica accessions collected throughout Vietnam and Laos.

Pennywort (Centella asiatica L.) is commonly grown in the tropical world for its nutritional and medicinal values. Valuable saponins in pennywort are extensively investigated for their anti-tumour activities. The diversity in morphology, phytochemical contents and genetics among pennywort accessions has been extensively studied to identify elite landraces for large-scale production. While pennywort is widely consumed in Vietnam, a systematic characterization of their diverse morphology, secondary metabolites and genetics is lacking. In this work, 26 pennywort accessions were collected across Vietnam and Laos. Their morphological features and yields were characterized under uniform agro-climatic conditions at Hue city in central Vietnam. The highest yield was obtained with HUIB_CA20 (478 g per tray), compared to the lowest yield in HUIB_CA19 (107 g per tray). Furthermore, a range of phytochemical markers, including vitamin C, reducing sugar, carotenoid, tannin, phenolic, flavonoid and saponin contents, were determined. Based on yield, phenolic and flavonoid contents, HUIB_CA20 and HUIB_CA27 were determined to be elite cultivars in this germplasm. Finally, microsatellite analysis was performed to explore the genetic diversity within the germplasm. Using fourteen SSR primer pairs, a total of 47 alleles were identified with 45 alleles (96 %) being polymorphic. These results will be useful for breeding programs aiming to create elite pennywort cultivars with enhanced properties.

Method to generate Holliday junction recombination intermediates via RecA-mediated four-strand exchange.

DNA molecules that contain single Holliday junctions have served as model substrates to investigate the pathway in which homologous recombination intermediates are processed. However, the preparation of DNA containing Holliday junctions in high yield remains a challenge. In this work, we used a nicking endonuclease to generate gapped DNA, from which α-structured DNA or figure-8 DNA were created via RecA-mediated reactions. The resulting DNA molecules were found to serve as good substrates for Holliday junction resolvases. The simplified method negates the requirement for radioactive labelling of DNA, making the generation of Holliday junction DNA more accessible to non-experts.

Single-molecule imaging reveals molecular coupling between transcription and DNA repair machinery in live cells.

The Escherichia coli transcription-repair coupling factor Mfd displaces stalled RNA polymerase and delivers the stall site to the nucleotide excision repair factors UvrAB for damage detection. Whether this handoff from RNA polymerase to UvrA occurs via the Mfd-UvrA2-UvrB complex or alternate reaction intermediates in cells remains unclear. Here, we visualise Mfd in actively growing cells and determine the catalytic requirements for faithful recruitment of nucleotide excision repair proteins. We find that ATP hydrolysis by UvrA governs formation and disassembly of the Mfd-UvrA2 complex. Further, Mfd-UvrA2-UvrB complexes formed by UvrB mutants deficient in DNA loading and damage recognition are impaired in successful handoff. Our single-molecule dissection of interactions of Mfd with its partner proteins inside live cells shows that the dissociation of Mfd is tightly coupled to successful loading of UvrB, providing a mechanism via which loading of UvrB occurs in a strand-specific manner.

Single-molecule live-cell imaging visualizes parallel pathways of prokaryotic nucleotide excision repair.

In the model organism Escherichia coli, helix distorting lesions are recognized by the UvrAB damage surveillance complex in the global genomic nucleotide excision repair pathway (GGR). Alternately, during transcription-coupled repair (TCR), UvrA is recruited to Mfd at sites of RNA polymerases stalled by lesions. Ultimately, damage recognition is mediated by UvrA, followed by verification by UvrB. Here we characterize the differences in the kinetics of interactions of UvrA with Mfd and UvrB by following functional, fluorescently tagged UvrA molecules in live TCR-deficient or wild-type cells. The lifetimes of UvrA in Mfd-dependent or Mfd-independent interactions in the absence of exogenous DNA damage are comparable in live cells, and are governed by UvrB. Upon UV irradiation, the lifetimes of UvrA strongly depended on, and matched those of Mfd. Overall, we illustrate a non-perturbative, imaging-based approach to quantify the kinetic signatures of damage recognition enzymes participating in multiple pathways in cells.