A Two-color Single-molecule Sequencing Platform and Its Clinical Applications.

DNA sequencers have become increasingly important research and diagnostic tools over the past 20 years. In this study, we developed a single-molecule desktop sequencer, GenoCare 1600 (GenoCare), which utilizes amplification-free library preparation and two-color sequencing-by-synthesis chemistry, making it more user-friendly compared with previous single-molecule sequencing platforms for clinical use. Using the GenoCare platform, we sequenced an Escherichia coli standard sample and achieved a consensus accuracy exceeding 99.99%. We also evaluated the sequencing performance of this platform in microbial mixtures and coronavirus disease 2019 (COVID-19) samples from throat swabs. Our findings indicate that the GenoCare platform allows for microbial quantitation, sensitive identification of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) virus, and accurate detection of virus mutations, as confirmed by Sanger sequencing, demonstrating its remarkable potential in clinical application.

A nonsynonymous mutation in BhLS, encoding an acyl-CoA N-acyltransferase leads to fruit and seed size variation in wax gourd (Benincasa hispida).

Wax gourd (Benincasa hispida (Thunb.) Cogn., 2n = 2x = 24) is an economically important vegetable crop cultivated widely in many tropical and subtropical regions, including China, India, and Japan. Both fruit and seeds are prized agronomic attributes in wax gourd breeding and production. However, the genetic mechanisms underlying these traits remain largely unexplored. In this study, we observed a strong correlation between fruit size and seed size variation in our mapping population, indicating genetic control by a single gene, BhLS, with large size being dominant over small. Through bulk segregant analysis sequencing and fine mapping with a large F2 population, we precisely located the BhLS gene within a 47.098-kb physical interval on Chromosome 10. Within this interval, only one gene, Bhi10M000649, was identified, showing homology to Arabidopsis HOOKLESS1. A nonsynonymous mutation (G to C) in the second exon of Bhi10M000649 was found to be significantly associated with both fruit and seed size variation in wax gourd. These findings collectively highlight the pleiotropic effect of the BhLS gene in regulating fruit and seed size in wax gourd. Our results offer molecular insights into the variation of fruit and seed size in wax gourd and establish a fundamental framework for breeding wax gourd cultivars with desired traits.

A transcriptome analysis of Benincasa hispida revealed the pathways and genes involved in response to Phytophthora melonis infection.

Wilt disease caused by Phytophthora melonis infection is one of the most serious threats to Benincasa hispida production. However, the mechanism of the response of B. hispida to a P. melonis infection remains largely unknown. In the present study, two B. hispida cultivars with different degrees of resistance to P. melonis were identified: B488 (a moderately resistant cultivar) and B214 (a moderately susceptible cultivar). RNA-seq was performed on P. melonis-infected B488 and B214 12 hours post infection (hpi). Compared with the control, 680 and 988 DEGs were respectively detected in B488 and B214. A KEGG pathway analysis combined with a cluster analysis revealed that phenylpropanoid biosynthesis, plant-pathogen interaction, the MAPK signaling pathway-plant, and plant hormone signal transduction were the most relevant pathways during the response of both B488 and B214 to P. melonis infection, as well as the differentially expressed genes in the two cultivars. In addition, a cluster analysis of transcription factor genes in DEGs identified four genes upregulated in B488 but not in B214 at 6 hpi and 12 hpi, which was confirmed by qRT-PCR. These were candidate genes for elucidating the mechanism of the B. hispida response to P. melonis infection and laying the foundation for the improvement of B. hispida.

A conserved double-W box in the promoter of CaWRKY40 mediates autoregulation during response to pathogen attack and heat stress in pepper.

CaWRKY40 was previously found to be transcriptionally up-regulated by Ralstonia solanacearum inoculation (RSI) or heat stress (HS), but the underlying mechanism remains unknown. Herein, we report that a double-W box-element (DWE) in the promoter of CaWRKY40 is critical for these responses. The upstream W box unit WI of this composite element is crucial for preferential binding by CaWRKY40 and responsiveness to RSI or HS. DWE-driven CaWRKY40 can be transcriptionally and nonspecifically regulated by itself and by CaWRKY58 and CaWRKY27. The DWE was also found in the promoters of CaWRKY40 orthologs, including AtWRKY40, VvWRKY40, GmWRKY40, CplWRKY40, SaWRKY40, SpWRKY40, NtWRKY40, and NaWRKY40. DWEAtWRKY40 was analogous to DWECaWRKY40 by responding to RSI or HS and AtWRKY40 expression. These data suggest that a conserved response of plants to pathogen infection or HS is probably mediated by binding of the DWE by WRKY40.

Ca14-3-3 Interacts With CaWRKY58 to Positively Modulate Pepper Response to Low-Phosphorus Starvation.

Low-phosphorus stress (LPS) and pathogen attack are two important stresses frequently experienced by plants in their natural habitats, but how plant respond to them coordinately remains under-investigated. Here, we demonstrate that CaWRKY58, a known negative regulator of the pepper (Capsicum annuum) response to attack by Ralstonia solanacearum, is upregulated by LPS. Virus-induced gene silencing (VIGS) and overexpression of CaWRKY58 in Nicotiana benthamiana plants in combination with chromatin immunoprecipitation (ChIP) and electrophoretic mobility shift assays (EMSA) demonstrated that CaWRKY58 positively regulates the response of pepper to LPS by directly targeting and regulating genes related to phosphorus-deficiency tolerance, including PHOSPHATE STARVATION RESPONSE1 (PHR1). Yeast two-hybrid assays revealed that CaWRKY58 interacts with a 14-3-3 protein (Ca14-3-3); this interaction was confirmed by pull-down, bimolecular fluorescence complementation (BiFC), and microscale thermophoresis (MST) assays. The interaction between Ca14-3-3 and CaWRKY58 enhanced the activation of PHR1 expression by CaWRKY58, but did not affect the expression of the immunity-related genes CaNPR1 and CaDEF1, which are negatively regulated by CaWRKY58 in pepper upon Ralstonia solanacearum inoculation. Collectively, our data indicate that CaWRKY58 negatively regulates immunity against Ralstonia solanacearum, but positively regulates tolerance to LPS and that Ca14-3-3 transcriptionally activates CaWRKY58 in response to LPS.

CaC3H14 encoding a tandem CCCH zinc finger protein is directly targeted by CaWRKY40 and positively regulates the response of pepper to inoculation by Ralstonia solanacearum.

Tandem CCCH zinc finger (TZnF) proteins have been implicated in plant defence, but their role in pepper (Capsicum annuum) is unclear. In the present study, the role of CaC3H14, a pepper TZnF protein, in the immune response of pepper plants to Ralstonia solanacearum infection was characterized. When fused to the green fluorescent protein, CaC3H14 was localized exclusively to the nuclei in leaf cells of Nicotiana benthamiana plants transiently overexpressing CaC3H14. Transcript abundance of CaC3H14 was up-regulated by inoculation with R. solanacearum. Virus-induced silencing of CaC3H14 increased the susceptibility of the plants to R. solanacearum and down-regulated the genes associated with the hypersensitive response (HR), specifically HIR1 and salicylic acid (SA)-dependent PR1a. By contrast, silencing resulted in the up-regulation of jasmonic acid (JA)-dependent DEF1 and ethylene (ET) biosynthesis-associated ACO1. Transient overexpression of CaC3H14 in pepper triggered an intensive HR, indicated by cell death and hydrogen peroxide (H2 O2 ) accumulation, up-regulated PR1a and down-regulated DEF1 and ACO1. Ectopic overexpression of CaC3H14 in tobacco plants significantly decreased the susceptibility of tobacco plants to R. solanacearum. It also up-regulated HR-associated HSR515, immunity-associated GST1 and the SA-dependent marker genes NPR1 and PR2, but down-regulated JA-dependent PR1b and ET-dependent EFE26. The CaC3H14 promoter and was bound and its transcription was up-regulated by CaWRKY40. Collectively, these results indicate that CaC3H14 is transcriptionally targeted by CaWRKY40, is a modulator of the antagonistic interaction between SA and JA/ET signalling, and enhances the defence response of pepper plants to infection by R. solanacearum.

Single molecule sequencing of the M13 virus genome without amplification.

Next generation sequencing (NGS) has revolutionized life sciences research. However, GC bias and costly, time-intensive library preparation make NGS an ill fit for increasing sequencing demands in the clinic. A new class of third-generation sequencing platforms has arrived to meet this need, capable of directly measuring DNA and RNA sequences at the single-molecule level without amplification. Here, we use the new GenoCare single-molecule sequencing platform from Direct Genomics to sequence the genome of the M13 virus. Our platform detects single-molecule fluorescence by total internal reflection microscopy, with sequencing-by-synthesis chemistry. We sequenced the genome of M13 to a depth of 316x, with 100% coverage. We determined a consensus sequence accuracy of 100%. In contrast to GC bias inherent to NGS results, we demonstrated that our single-molecule sequencing method yields minimal GC bias.

Single molecule targeted sequencing for cancer gene mutation detection.

With the rapid decline in cost of sequencing, it is now affordable to examine multiple genes in a single disease-targeted clinical test using next generation sequencing. Current targeted sequencing methods require a separate step of targeted capture enrichment during sample preparation before sequencing. Although there are fast sample preparation methods available in market, the library preparation process is still relatively complicated for physicians to use routinely. Here, we introduced an amplification-free Single Molecule Targeted Sequencing (SMTS) technology, which combined targeted capture and sequencing in one step. We demonstrated that this technology can detect low-frequency mutations using artificially synthesized DNA sample. SMTS has several potential advantages, including simple sample preparation thus no biases and errors are introduced by PCR reaction. SMTS has the potential to be an easy and quick sequencing technology for clinical diagnosis such as cancer gene mutation detection, infectious disease detection, inherited condition screening and noninvasive prenatal diagnosis.

Genome-wide identification and transcriptional expression analysis of mitogen-activated protein kinase and mitogen-activated protein kinase kinase genes in Capsicum annuum.

The tripartite mitogen-activated protein kinase (MAPK) signaling cascades have been implicated in plant growth, development, and environment adaptation, but a comprehensive understanding of MAPK signaling at genome-wide level is limited in Capsicum annuum. Herein, genome-wide identification and transcriptional expression analysis of MAPK and MAPK kinase (MAPKK) were performed in pepper. A total of 19 pepper MAPK (CaMAPKs) genes and five MAPKK (CaMAPKKs) genes were identified. Phylogenetic analysis indicated that CaMAPKs and CaMAPKKs could be classified into four groups and each group contains similar exon-intron structures. However, significant divergences were also found. Notably, five members of the pepper MAPKK family were much less conserved than those found in Arabidopsis, and 9 Arabidopsis MAPKs did not have orthologs in pepper. Additionally, 7 MAPKs in Arabidopsis had either two or three orthologs in the pepper genome, and six pepper MAPKs and one MAPKK differing in sequence were found in three pepper varieties. Quantitative real-time RT-PCR analysis showed that the majority of MAPK and MAPKK genes were ubiquitously expressed and transcriptionally modified in pepper leaves after treatments with heat, salt, and Ralstonia solanacearum inoculation as well as exogenously applied salicylic acid, methyl jasmonate, ethephon, and abscisic acid. The MAPKK-MAPK interactome was tested by yeast two-hybrid assay, the results showed that one MAPKK might interact with multiple MAPKs, one MAPK might also interact with more than one MAPKKs, constituting MAPK signaling networks which may collaborate in transmitting upstream signals into appropriate downstream cellular responses and processes. These results will facilitate future functional characterization of MAPK cascades in pepper.

SRC2-1 is required in PcINF1-induced pepper immunity by acting as an interacting partner of PcINF1.

Elicitins are elicitors that can trigger hypersensitive cell death in most Nicotiana spp., but their underlying molecular mechanism is not well understood. The gene Phytophthora capsici INF1 (PcINF1) coding for an elicitin from P. capsici was characterized in this study. Transient overexpression of PcINF1 triggered cell death in pepper (Capsicum annuum L.) and was accompanied by upregulation of the hypersensitive response marker, Hypersensitive Induced Reaction gene 1 (HIR1), and the pathogenesis-related genes SAR82, DEF1, BPR1, and PO2. A putative PcINF1-interacting protein, SRC2-1, was isolated from a pepper cDNA library by yeast two-hybrid screening and was observed to target the plasma membrane. The interaction between PcINF1 and SRC2-1 was confirmed by bimolecular fluorescence complementation and co-immunoprecipitation. Simultaneous transient overexpression of SRC2-1 and PcINF1 in pepper plants triggered intensive cell death, whereas silencing of SRC2-1 by virus-induced gene silencing blocked the cell death induction of PcINF1 and increased the susceptibility of pepper plants to P. capsici infection. Additionally, membrane targeting of the PcINF1-SRC2-1 complex was required for cell death induction. The C2 domain of SRC2-1 was crucial for SRC2-1 plasma membrane targeting and the PcINF1-SRC2-1 interaction. These results suggest that SRC2-1 interacts with PcINF1 and is required in PcINF1-induced pepper immunity.