Development of a reverse transcription loop-mediated isothermal amplification assay with novel quantitative pH biosensor readout method for SARS-CoV-2 detection.

Reverse transcription loop-mediated isothermal amplification (RT-LAMP) is a molecular amplification method that can detect SARS-CoV-2 in a shorter time than the current gold-standard molecular diagnostic reverse transcription-polymerase chain reaction (RT-PCR). However, previously developed RT-LAMP assays have mostly relied on highly subjective visual colorimetric interpretation. In this study, an RT-LAMP assay was developed with quantitative measurement of reaction pH using a novel portable pH biosensor compared to qualitative colorimetric interpretation and gel electrophoresis, with 57 clinical COVID-19 samples used for validation of the test. The LoD of the assay is 103 copies/µL. The highest sensitivity was found in the qualitative methods (93.75%), while the highest specificity and likelihood ratio was found in the pH sensor (87.5% and 6.72). On the sensor measurement, a significant difference (p < 0.0001) was observed between the average pH of the RT-PCR (+) COVID-19 (6.15 ± 0.27), while the average pH of the RT-PCR (-) samples (6.72 ± 0.22). Correlation analysis revealed a strong correlation (r = 0.78, p < 0.0001) between the Ct values obtained from RT-PCR with the biosensor pH readout. RT-LAMP with the quantitative pH sensor readout method has the potential to be further developed as an objective molecular assay for rapid and simple detection of SARS-CoV-2.

Salt tolerance QTLs of an endemic rice landrace, Horkuch at seedling and reproductive stages.

Salinity has a significant negative impact on production of rice. To cope with the increased soil salinity due to climate change, we need to develop salt tolerant rice varieties that can maintain their high yield. Rice landraces indigenous to coastal Bangladesh can be a great resource to study the genetic basis of salt adaptation. In this study, we implemented a QTL analysis framework with a reciprocal mapping population developed from a salt tolerant landrace Horkuch and a high yielding rice variety IR29. Our aim was to detect genetic loci that contributes to the salt adaptive responses of the two different developmental stages of rice which are very sensitive to salinity stress. We identified 14 QTLs for 9 traits and found that most are unique to specific developmental stages. In addition, we detected a significant effect of the cytoplasmic genome on the QTL model for some traits such as leaf total potassium and filled grain weight. This underscores the importance of considering cytoplasm-nuclear interaction for breeding programs. Finally, we identified QTLs co-localization for multiple traits that highlights the possible constraint of multiple QTL selection for breeding programs due to different contributions of a donor allele for different traits.

Functional genomic analysis of K+ related salt-responsive transporters in tolerant and sensitive genotypes of rice.

Introduction: Salinity is a complex environmental stress that affects the growth and production of rice worldwide. But there are some rice landraces in coastal regions that can survive in presence of highly saline conditions. An understanding of the molecular attributes contributing to the salinity tolerance of these genotypes is important for developing salt-tolerant high yielding modern genotypes to ensure food security. Therefore, we investigated the role and functional differences of two K+ salt-responsive transporters. These are OsTPKa or Vacuolar two-pore potassium channel and OsHAK_like or a hypothetical protein of the HAK family. These transporters were selected from previously identified QTLs from the tolerant rice landrace genotype (Horkuch) and sensitive genotype (IR29). Methods: In silico comparative sequence analysis of the promoter sequences of two these genes between Horkuch and IR29 was done. Real-Time expression of the selected genes in leaves and roots of IR29 (salt-sensitive), I-14 and I-71 (Recombinant Inbred Lines of IR29(♀)× Horkuch), Horkuch and Pokkali (salt-tolerant) under salt-stress at different time points was analyzed. For further insight, OsTPKa and OsHAK_like were chosen for loss-of-function genomic analysis in Horkuch using the CRISPR/Cas9 tool. Furthermore, OsTPKa was chosen for cloning into a sensitive variety by Gateway technology to observe the effect of gain-of-function. Results: The promoter sequences of the OsTPKa and OsHAK_like genes showed some significant differences in promoter sequences which may give a survival advantage to Horkuch under salt-stress. These two genes were also found to be overexpressed in tolerant varieties (Horkuch and Pokkali). Moreover, a coordinated expression pattern between these two genes was observed in tolerant Horkuch under salt-stress. Independently transformed plants where the expression of these genes was significantly lowered, performed poorly in physiological tests for salinity tolerance. On the other hand, positively transformed T0 plants with the OsTPKa gene from Horkuch consistently showed growth advantage under both control and salt stress. Discussion: The poor performance of the transgenic plants with the down-regulated genes OsTPKa and OsHAK_like under salt stress supports the assumption that OsTPKa and OsHAK_like play important roles in defending the rice landrace Horkuch against salt stress, minimizing salt injury, and maintaining plant growth. Moreover, the growth advantage provided by overexpression of the vacuolar OsTPKa K+ transporter, particularly under salt stress reconfirms its important role in providing salt tolerance. The QTL locus from Horkuch containing these two transporters maybe bred into commercial rice to produce high-yielding salt tolerant rice.

Pseudomonas aeruginosa PA80 is a cystic fibrosis isolate deficient in RhlRI quorum sensing.

Pseudomonas aeruginosa uses quorum sensing (QS) to modulate the expression of several virulence factors that enable it to establish severe infections. The QS system in P. aeruginosa is complex, intricate and is dominated by two main N-acyl-homoserine lactone circuits, LasRI and RhlRI. These two QS systems work in a hierarchical fashion with LasRI at the top, directly regulating RhlRI. Together these QS circuits regulate several virulence associated genes, metabolites, and enzymes in P. aeruginosa. Paradoxically, LasR mutants are frequently isolated from chronic P. aeruginosa infections, typically among cystic fibrosis (CF) patients. This suggests P. aeruginosa can undergo significant evolutionary pathoadaptation to persist in long term chronic infections. In contrast, mutations in the RhlRI system are less common. Here, we have isolated a clinical strain of P. aeruginosa from a CF patient that has deleted the transcriptional regulator RhlR entirely. Whole genome sequencing shows the rhlR locus is deleted in PA80 alongside a few non-synonymous mutations in virulence factors including protease lasA and rhamnolipid rhlA, rhlB, rhlC. Importantly we did not observe any mutations in the LasRI QS system. PA80 does not appear to have an accumulation of mutations typically associated with several hallmark pathoadaptive genes (i.e., mexT, mucA, algR, rpoN, exsS, ampR). Whole genome comparisons show that P. aeruginosa strain PA80 is closely related to the hypervirulent Liverpool epidemic strain (LES) LESB58. PA80 also contains several genomic islands (GI's) encoding virulence and/or resistance determinants homologous to LESB58. To further understand the effect of these mutations in PA80 QS regulatory and virulence associated genes, we compared transcriptional expression of genes and phenotypic effects with isogenic mutants in the genetic reference strain PAO1. In PAO1, we show that deletion of rhlR has a much more significant impact on the expression of a wide range of virulence associated factors rather than deletion of lasR. In PA80, no QS regulatory genes were expressed, which we attribute to the inactivation of the RhlRI QS system by deletion of rhlR and mutation of rhlI. This study demonstrates that inactivation of the LasRI system does not impact RhlRI regulated virulence factors. PA80 has bypassed the common pathoadaptive mutations observed in LasR by targeting the RhlRI system. This suggests that RhlRI is a significant target for the long-term persistence of P. aeruginosa in chronic CF patients. This raises important questions in targeting QS systems for therapeutic interventions.

Gene Expression analysis associated with salt stress in a reciprocally crossed rice population.

The rice landrace Horkuch, endemic to the southern saline coast of Bangladesh, is known to have salt tolerance traits and can therefore contribute to a high yielding recipient for breeding purposes. In this study, we reciprocally crossed Horkuch with high yielding but salt sensitive IR29 to detect the complement of genes that were responsible for conferring salt tolerance versus sensitivity at the seedling developmental stage. We looked at tolerant and sensitive F3 families from individual F2 segregating plants and analyzed them for differential gene expressions using RNAseq. In general, we observed higher numbers of genes differentially expressed in leaves compared to root tissues. This included both upregulation and downregulation of gene expression across our experimental factors. Gene expression decreased in sensitive leaf after stress exposure where tolerant plants showed the opposite trend. In root, tolerant plants expression decreased at higher time points of stress exposure. We also observed a strong maternal cytoplasmic effect on gene expression and this was most evident in roots where there was upregulation in functional enrichments related to phosphorylation, electron carriers, transporter and cation transmembrane activities. Stress groups (tolerant and sensitive) response in F3 families were distinctive in both cytoplasmic backgrounds and involved uniquely upregulated genes in tolerant progenies including membrane sensor proteins, enzymes involved with signaling pathways, such as those producing trehalose and G-protein coupled receptor proteins, photosynthesis-related enzymes and golgi body recycling as well as prolamin precursor proteins involved in refolding of proteins. On the other hand, sensitivity was found to be associated with differential upregulation of only a few redox proteins and higher number of apoptosis related genes compared to the tolerant response. Overall, our highly replicated experimental design was powerful and allowed the detection of relatively subtle differential expression. Our future goal is to correlate these expression differences with QTLs in this population, which would help identify the relative importance of specific genetic loci and provide a direct avenue for combining higher levels of salt tolerance with better agronomic traits in rice.

Reproductive stage physiological and transcriptional responses to salinity stress in reciprocal populations derived from tolerant (Horkuch) and susceptible (IR29) rice.

Global increase in salinity levels has made it imperative to identify novel sources of genetic variation for tolerance traits, especially in rice. The rice landrace Horkuch, endemic to the saline coastal area of Bangladesh, was used in this study as the source of tolerance in reciprocal crosses with the sensitive but high-yielding IR29 variety for discovering transcriptional variation associated with salt tolerance in the resulting populations. The cytoplasmic effect of the Horkuch background in leaves under stress showed functional enrichment for signal transduction, DNA-dependent regulation and transport activities. In roots the enrichment was for cell wall organization and macromolecule biosynthesis. In contrast, the cytoplasmic effect of IR29 showed upregulation of apoptosis and downregulation of phosphorylation across tissues relative to Horkuch. Differential gene expression in leaves of the sensitive population showed downregulation of GO processes like photosynthesis, ATP biosynthesis and ion transport. Roots of the tolerant plants conversely showed upregulation of GO terms like G-protein coupled receptor pathway, membrane potential and cation transport. Furthermore, genes involved in regulating membrane potentials were constitutively expressed only in the roots of tolerant individuals. Overall our work has developed genetic resources and elucidated the likely mechanisms associated with the tolerance response of the Horkuch genotype.

Comparative genomics of two jute species and insight into fibre biogenesis.

Jute (Corchorus sp.) is one of the most important sources of natural fibre, covering ∼80% of global bast fibre production1. Only Corchorus olitorius and Corchorus capsularis are commercially cultivated, though there are more than 100 Corchorus species2 in the Malvaceae family. Here we describe high-quality draft genomes of these two species and their comparisons at the functional genomics level to support tailor-designed breeding. The assemblies cover 91.6% and 82.2% of the estimated genome sizes for C. olitorius and C. capsularis, respectively. In total, 37,031 C. olitorius and 30,096 C. capsularis genes are identified, and most of the genes are validated by cDNA and RNA-seq data. Analyses of clustered gene families and gene collinearity show that jute underwent shared whole-genome duplication ∼18.66 million years (Myr) ago prior to speciation. RNA expression analysis from isolated fibre cells reveals the key regulatory and structural genes involved in fibre formation. This work expands our understanding of the molecular basis of fibre formation laying the foundation for the genetic improvement of jute.

Enhanced Salt Tolerance Conferred by the Complete 2.3 kb cDNA of the Rice Vacuolar Na(+)/H(+) Antiporter Gene Compared to 1.9 kb Coding Region with 5' UTR in Transgenic Lines of Rice.

Soil salinity is one of the most challenging problems that restricts the normal growth and production of rice worldwide. It has therefore become very important to produce more saline tolerant rice varieties. This study shows constitutive over-expression of the vacuolar Na(+)/H(+) antiporter gene (OsNHX1) from the rice landrace (Pokkali) and attainment of enhanced level of salinity tolerance in transgenic rice plants. It also shows that inclusion of the complete un-translated regions (UTRs) of the alternatively spliced OsNHX1 gene provides a higher level of tolerance to the transgenic rice. Two separate transformation events of the OsNHX1 gene, one with 1.9 kb region containing the 5' UTR with CDS and the other of 2.3 kb, including 5' UTR, CDS, and the 3' UTR regions were performed. The transgenic plants with these two different constructs were advanced to the T3 generation and physiological and molecular screening of homozygous plants was conducted at seedling and reproductive stages under salinity (NaCl) stress. Both transgenic lines were observed to be tolerant compared to WT plants at both physiological stages. However, the transgenic lines containing the CDS with both the 5' and 3' UTR were significantly more tolerant compared to the transgenic lines containing OsNHX1 gene without the 3' UTR. At the seedling stage at 12 dS/m stress, the chlorophyll content was significantly higher (P < 0.05) and the electrolyte leakage significantly lower (P < 0.05) in the order 2.3 kb > 1.9 kb > and WT lines. Yield in g/plant in the best line from the 2.3 kb plants was significantly more (P < 0.01) compared, respectively, to the best 1.9 kb line and WT plants at stress of 6 dS/m. Transformation with the complete transcripts rather than the CDS may therefore provide more durable level of tolerance.

Unique Genotypic Differences Discovered among Indigenous Bangladeshi Rice Landraces.

Bangladesh is a reservoir of diverse rice germplasm and is home to many landraces with unique, important traits. Molecular characterization of these landraces is of value for their identification, preservation, and potential use in breeding programs. Thirty-eight rice landraces from different regions of Bangladesh including some high yielding BRRI varieties were analyzed by 34 polymorphic microsatellite markers yielding a total of 258 reproducible alleles. The analysis could locate 34 unique identifiers for 21 genotypes, making the latter potentially amenable to identity verification. An identity map for these genotypes was constructed with all the 12 chromosomes of the rice genome. Polymorphism information content (PIC) scores of the 34 SSR markers were 0.098 to 0.89 where on average 7.5 alleles were observed. A dendogram constructed using UPGMA clustered the varieties into two major groups and five subgroups. In some cases, the clustering matched with properties like aromaticity, stickiness, salt tolerance, and photoperiod insensitivity. The results will help breeders to work towards the proper utilization of these landraces for parental selection and linkage map construction for discovery of useful alleles.

Physiology and gene expression of the rice landrace Horkuch under salt stress.

Good donors in breeding for salt tolerance are a prerequisite for food security under changing climatic conditions. Horkuch, a farmer-popular salt tolerant rice (Oryza sativa L.) variety from the south-west coast of Bangladesh was characterised up to maturity under NaCl stress, together with a modern variety (BRRI dhan41), a sensitive control (BRRI dhan29) and Pokkali, the salt-tolerant benchmark for rice. Horkuch had low reduction in shoot biomass, a low Na:K ratio in flag leaves, a low percent reduction in yield and good partitioning of Na in the older leaves, and maintained high levels of Ca and Mg in the flag leaves. In order to understand the physiology at the molecular level, the expression of salt-responsive genes was investigated using microarray analysis. Salt-stressed cDNA of Horkuch seedlings were hybridised with cDNA probes synthesised mainly from database sequences of Arabidopsis thaliana (L.) Heynh. The upregulated genes included transcription factors, signal transducers, metabolic enzymes, reactive oxygen species (ROS) scavengers, osmoprotectants and some specific salt-induced transcripts. An increase in expression of photosynthesis-related genes as well ROS scavengers suggested that this could be the reason for the better yield performance of Horkuch. The data therefore indicate Horkuch as a potential donor alternative to Pokkali in breeding programs for salt tolerance.