Arabidopsis transcriptome dataset of the response of imbibed wild-type and glucosinolate-deficient seeds to nitrogen-containing compounds.

The presented RNAseq data were obtained from Arabidopsis seeds dry and 6h imbibed to describe, in wild-type and glucosinolate (GSL)-deficient genotypes, the response at the RNA level to nitrogen compounds, i.e., potassium nitrate (KNO3, 10mM), potassium thiocyanate (KSCN, 8µM). The cyp79B2 cyp79B3 (cyp79B2/B3) double mutant deficient in Indole GSL, the myb28 myb29 (myb28/29) double mutant deficient in aliphatic GSL, the quadruple mutant cyp79B2 cyp79B3 myb28 myb29 (qko) deficient in total GSL in the seed and the WT reference genotype in Col-0 background were used for the transcriptomic analysis. Total ARN was extracted using NucleoSpin® RNA Plant and Fungi kit. Library construction and sequencing were performed with DNBseq™ technology at Beijing Genomics Institute. FastQC was used to check reads quality and mapping analysis were made using a quasi-mapping alignment from Salmon. Gene expression changes in mutant seeds compared to WT were calculated using DESeq2 algorithms. This comparison with the qko, cyp79B2/B3 and myb28/29 mutants made it possible to identify 30220, 36885 and 23807 differentially expressed genes (DEGs), respectively. Mapping rate result was merge into a single report using MultiQC; graphic results were illustrated through Veen diagrams and volcano plots. Fastq raw data and count files from 45 samples are available in the repository Sequence Read Archive (SRA) of the National Center for Biotechnology Information (NCBI) and can be consulted with the data identification number GSE221567 at https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE221567.

Surveys in the Chrysanthemum Production Areas of Brazil and Colombia Reveal That Weeds Are Potential Reservoirs of Chrysanthemum Stunt Viroid.

The stunting disease, incited by chrysanthemum stunt viroid (CSVd), has become a serious problem in chrysanthemum production areas worldwide. Here we identified 46 weed species from chrysanthemum fields in two producing regions of the State of São Paulo, Brazil. The mechanical inoculation of these weeds with a Brazilian CSVd isolate revealed that this viroid was able to infect 17 of these species, in addition to chrysanthemum, tomato and potato. Plants of Oxalis latifolia and chrysanthemum naturally infected with CSVd were found in chrysanthemum fields in Colombia, which is the first CSVd report in that country. Therefore, weeds have the potential to act as reservoirs of CSVd in the field. These results are the first reports of experimental CSVd infection in the following species: Amaranthus viridis, Cardamine bonariensis, Chamaesyce hirta, Conyza bonariensis, Digitaria sanguinalis, Gomphrena globosa, Helianthus annuus, Lupinus polyphyllus, Mirabilis jalapa, Oxalis latifolia, Portulaca oleracea and Catharanthus roseus. The phylogenetic analyses of the CSVd variants identified herein showed three groups with Brazilian CSVd variants distributed in them all, which suggests that Brazilian CSVd isolates may have different origins through successive introductions of infected germplasm of chrysanthemum in Brazil.

Duplex RT-PCR assay for simultaneous detection of TSWV and CSVd in chrysanthemum.

A novel duplex RT-PCR assay for simultaneous detection of TSWV and CSVd in chrysanthemums was developed. Previous reported primers for amplification of TSWV and CSVd were used and a novel pair of primers for CSVd was designed to improve duplex amplification compatibility. Sensitivity and efficiency of the previous reported and novel primers for CSVd were assessed. Then, the sensitivity of the combined primers to amplify both TSWV and CSVd cDNA were also evaluated. Both TSWV and CSVd were detected in preparations diluted up to 10-4 and 10-5 respectively, from total RNA extracts. This duplex RT-PCR method showed an estimated diagnostic sensitivity (DSe) of 97% and diagnostic specificity (DSp) of 99%. For combination of the primers TSWV L1/ L2 and CSVd UCO-1 F/ UCO-1R, the protocol could detect pathogen RNA from naturally infected plants until 0.1 ng and 1 ng respectively. This novel protocol for detection of TSWV/CSVd represents a useful diagnostic tool without the need of expensive probes and less extensive laboratory work. This method could be helpful to assist the selection and further propagation of healthy chrysanthemums on the field as well as to understand the dynamics and the interaction of this virus and viroid within farms.

Differential disease symptoms and full-length genome sequence analysis for three strains of Tobacco etch virus.

Tobacco etch virus (TEV) strains HAT, Mex21, and N have been the focus of numerous studies to dissect a host resistance mechanism in Capsicum spp. Little is known, however, about their general pathogenicity and genomic sequence data are not available on the TEV strains Mex21 and N. Four Nicotiana spp. were evaluated after inoculation with each TEV strain. Nicotiana tabacum 'Kentucky 14' and N. clevelandii plants expressed varied systemic symptoms dependent on the TEV strain; however, disease severity increased from HAT (mild mosaic symptoms) to Mex21 (more severe mosaic symptoms with stunting) to N (severe chlorosis and stunting). Nicotiana tabacum 'Samsun' plants developed relatively milder symptoms and N. glutinosa plants remained symptomless, although they were systemically infected. The genome of each TEV strain was sequenced and shown to consist of 9,495 nucleotides and a polyprotein of 3,054 amino acids. Comparison of their nucleotide sequences relative to the original HAT sequence (GenBank Accession No. M11458) revealed 95, 92, and 92 % identity for HAT-AU (from Auburn University), Mex21, and N, respectively. HAT-AU had 91 % sequence identity with Mex21 and N, while Mex21 and N were more closely related with 98 % nucleotide sequence identity. Similarly, the amino acid sequence identities for the full-length polyprotein ranged from 95 % for HAT-AU when compared with N to a high of 98 % identity between Mex21 and N.

Electroporetic transfection of pepper protoplasts with plant potyviruses.

Potyviruses are a persistent threat to bell pepper (Capsicum annuum L.) production worldwide. Much effort has been expended to study the resistance response of pepper cultivars at whole plant levels but with only limited effort at the cellular level using protoplasts. A pepper protoplast isolation procedure is available but an inoculation procedure is needed that provides consistent and highly efficient infection. An electroporation-based procedure for inoculation of potyviruses was developed using a base procedure developed for Cucumber mosaic virus (CMV). The final parameters identified for efficient potyvirus infection of pepper protoplasts involves two 25ms pulses, 200V each pulse with a 10s interval between pulses. Depending on the method of detection, e.g., ELISA versus RT-PCR, potyvirus RNA inoculum ranged from 10 to 40µg with infection detection occurring with samples of 50,000-100,000 protoplasts.

Cucumber mosaic virus 2b-Deficient Mutant Causes Limited, Asymptomatic Infection of Bell Pepper.

Cucumber mosaic virus Fast New York strain (CMV-Fny) containing a mutated 2b protein (CMV-FnyΔ2b) was evaluated for the ability to infect 'Calwonder' bell pepper (Capsicum annuum) plants in comparative tests with the parent virus, CMV-Fny. Plants inoculated with CMV-FnyΔ2b did not develop local or systemic symptoms of infection, whereas CMV-Fny-infected plants developed systemic chlorosis by 7 days post inoculation (dpi), followed by mosaic and leaf deformation. Virus accumulation, determined by enzyme-linked immunosorbent assay (ELISA), revealed that CMV-FnyΔ2b accumulated in inoculated Calwonder leaves and inconsistently infected some noninoculated leaves at a low titer but was not detected in the youngest, noninoculated leaves. Immuno-tissue blot tests did not detect CMV-FnyΔ2b in the stems of infected plants, whereas CMV-Fny accumulated throughout the length of the stems of inoculated plants. In two experiments, protoplasts were isolated from Calwonder leaves, inoculated with viral RNAs of CMV-Fny or CMV-FnyΔ2b, and tested by ELISA for infection. In both experiments, less CMV-FnyΔ2b than CMV-Fny accumulated in protoplasts. These results suggest that the CMV 2b protein is needed for systemic infection of Calwonder pepper plants and for accumulation of the virus in inoculated protoplasts.