Xanthomonas strains isolated from hosts in the Araceae reveal diverse phylogenetic relationships and origins.

The Araceae family, comprising ornamentals including Anthurium, Dieffenbachia, Philodendron, Colocasia, and Zantedeschia, is susceptible to Xanthomonas pathogens. Previous analyses have established heterogeneity in aroid strains, yet unresolved taxonomic positions and dynamics between Xanthomonas and frequently associated Stenotrophomonas in aroids necessitate in-depth genetic investigation to resolve these complex relationships. This study utilized multi-locus sequence analysis (MLSA) of housekeeping genes atpD, dnaA, dnaK, gltA, and gyrB to investigate 59 aroid strains, selected based on hosts, time, and geographical origins. After adding sequences from additional strains from NCBI GenBank, analysis of 161 concatenated sequences indicated that all aroid strains fell within Xanthomonas and Stenotrophomonas. Thirty-six strains isolated from Anthurium grouped under X. phaseoli, with outliers including one strain each in X. arboricola and X. sacchari, and two in Stenotrophomonas. Six strains from Caladium, Dieffenbachia, and Philodendron formed host-specific subgroups within X. euvesicatoria. One strain from Dieffenbachia aligned with X. campestris, while strains from Colocasia, Aglaonema, and Spathiphyllum clustered with X. sacchari. Apart from the zantedeschia strain described as X. arboricola pv. zantedeschiae, two colocasia, one epipremnum, and one anthurium strain joined the X. arboricola group. Overall, this study revealed significant heterogeneity among aroid strains, with anthurium strains clustering closely despite distant geographical origins. The analysis underscores the complexity of host-pathogen specificity within Xanthomonas and emphasizes the need for further taxonomic clarification through whole genome analysis of representative strains. The finding of this research will facilitate strain selection for inclusivity and exclusivity panels in developing diagnostic assays for X. phaseoli and xanthomonads affecting aroids.

Organ-specific transcriptome profiling of metabolic and pigment biosynthesis pathways in the floral ornamental progenitor species Anthurium amnicola Dressler.

Anthurium amnicola Dressler possesses a number of desirable and novel ornamental traits such as a purple-colored upright spathe, profuse flowering, and floral scent, some of which have been introgressed into modern Anthurium cultivars. As a first step in identifying genes associated with these traits, the transcriptome from root, leaf, spathe, and spadix from an accession of A. amnicola was assembled, resulting in 28,019 putative transcripts representing 19,458 unigenes. Genes involved in pigmentation, including those for the metabolism of chlorophyll and the biosynthesis of carotenoids, phenylpropanoids, and flavonoids were identified. The expression levels of one MYB transcription factor was highly correlated with naringenin 3-dioxygenase (F3H) and dihydroflavonol-4-reductase (DFR) in leaves, whereas a bHLH transcription factor was highly correlated with flavonoid 3'-monooxygenase (F3'H) and a DFR in spathes, suggesting that these two transcription factors might regulate flavonoid and anthocyanin synthesis in A. amnicola. Gene sequence and expression data from four major organs of A. amnicola provide novel basal information for understanding the genetic bases of ornamental traits and the determinants and evolution of form and function in the Araceae.

CRISPRi-mediated metabolic engineering of E. coli for O-methylated anthocyanin production.

BACKGROUND: Anthocyanins are a class of brightly colored, glycosylated flavonoid pigments that imbue their flower and fruit host tissues with hues of predominantly red, orange, purple, and blue. Although all anthocyanins exhibit pH-responsive photochemical changes, distinct structural decorations on the core anthocyanin skeleton also cause dramatic color shifts, in addition to improved stabilities and unique pharmacological properties. In this work, we report for the first time the extension of the reconstituted plant anthocyanin pathway from (+)-catechin to O-methylated anthocyanins in a microbial production system, an effort which requires simultaneous co-option of the endogenous metabolites UDP-glucose and S-adenosyl-L-methionine (SAM or AdoMet). RESULTS: Anthocyanin O-methyltransferase (AOMT) orthologs from various plant sources were co-expressed in Escherichia coli with Petunia hybrida anthocyanidin synthase (PhANS) and Arabidopsis thaliana anthocyanidin 3-O-glucosyltransferase (At3GT). Vitis vinifera AOMT (VvAOMT1) and fragrant cyclamen 'Kaori-no-mai' AOMT (CkmOMT2) were found to be the most effective AOMTs for production of the 3'-O-methylated product peonidin 3-O-glucoside (P3G), attaining the highest titers at 2.4 and 2.7 mg/L, respectively. Following modulation of plasmid copy number and optimization of VvAOMT1 and CkmOMT2 expression conditions, production was further improved to 23 mg/L using VvAOMT1. Finally, CRISPRi was utilized to silence the transcriptional repressor MetJ in order to deregulate the methionine biosynthetic pathway and improve SAM availability for O-methylation of cyanidin 3-O-glucoside (C3G), the biosynthetic precursor to P3G. MetJ repression led to a final titer of 51 mg/L (56 mg/L upon scale-up to shake flask), representing a twofold improvement over the non-targeting CRISPRi control strain and 21-fold improvement overall. CONCLUSIONS: An E. coli strain was engineered for production of the specialty anthocyanin P3G using the abundant and comparatively inexpensive flavonol precursor, (+)-catechin. Furthermore, dCas9-mediated transcriptional repression of metJ alleviated a limiting SAM pool size, enhancing titers of the methylated anthocyanin product. While microbial production of P3G and other O-methylated anthocyanin pigments will likely be valuable to the food industry as natural food and beverage colorants, we expect that the strain constructed here will also prove useful to the ornamental plant industry as a platform for evaluating putative anthocyanin O-methyltransferases in pursuit of bespoke flower pigment compositions.