Transient expression analysis of promoters of okra enation leaf curl virus in Nicotiana benthamiana, cotton and okra plants.

Viral promoters can be used to drive heterologous gene expression in transgenic plants. As part of our quest to look for new promoters, we have explored, for the first time, the promoters of okra enation leaf curl virus (OELCuV), a begomovirus infecting okra (Abelmoschus esculentus). The Rep and CP promoters of OELCuV fused with the gfp reporter gene, were expressed transiently in the natural host okra and the laboratory host cotton and Nicotiana benthamiana. The expression levels of the promoters were quantified through confocal laser scanning microscopy and GFP assay in N. benthamiana and okra. The results indicated that the Rep promoter was more active than the CP promoter, whose activity was similar to that of CaMV 35S promoter. Additionally, the Rep and CP promoters showed increase of expression, probably due to transactivation, when assayed following inoculation of OELCuV and betasatellite DNAs in cotton plants. A moderate increase in promoter activity in N. benthamiana was also seen, when assayed following the inoculation of the heterologous begomovirus Sri Lankan cassava mosaic virus.

P4 protein of an Indian isolate of rice tungro bacilliform virus modulates gene silencing.

Plant hosts and their viral pathogens are engaged in a constant cycle of defense and counter-defense as part of a molecular arms race, principal among them being the plant RNAi defense and the viral RNAi suppressor counter-defense. Rice tungro bacilliform virus (RTBV), member of the family Caulimoviridae, genus Tungrovirus, species Tungrovirus oryzae, infects rice in South- and Southeast Asia and causes severe symptoms of stunting, yellow-orange discoloration and twisting of leaf tips. To better understand the possible counter-defensive roles of RTBV against the host RNAi defense system, we explored the ability of the P4 protein of an Indian isolate of RTBV to act as a possible modulator of RNAi. Using a transient silencing and silencing suppression assay in Nicotiana benthamiana, we show that P4 not only displays an RNAi suppressor function, but also potentially enhances RNAi. The results also suggests that the N-terminal 168 amino acid residues of P4 are sufficient to maintain RNAi suppressor activity. Taken together with the earlier reports this work strengthens the view that the P4 protein carries out RNAi suppressor and a potential RNAi enhancer function.

Silencing suppressor protein PRT of rice tungro bacilliform virus interacts with the plant RNA silencing-related protein SGS3.

BACKGROUND: Rice tungro bacilliform virus (RTBV) is a double stranded DNA containing virus which causes the devastating tungro disease of rice in association with an RNA virus, rice tungro spherical virus. RNA silencing is an evolutionarily conserved antiviral defence pathway in plants as well as in several classes of higher organisms. Many viruses, in turn, encode proteins which are termed Viral Suppressor of RNA Silencing (VSR) because they downregulate or suppress RNA silencing. RESULTS: Using an RNA silencing suppressor assay we show that RTBV protease (PRT) acts as a mild VSR. A truncated version of PRT gene abolished the silencing suppression activity. We also show in planta interaction of PRT with the SGS3 protein of Solanum tuberosum and Arabidopsis thaliana using bimolecular fluorescence complementation assay (BIFC). Transient expression of PRT in Nicotiana benthamiana caused an increased accumulation of the begomovirus Sri Lankan cassava mosaic virus (SLCMV) DNA-A, which indicated a virulence function imparted on an unrelated virus. CONCLUSION: The finding supports the idea that PRT acts as suppressor of RNA silencing and this action may be mediated by its interaction with SGS3.

Characterization of the functional domains of nuclear shuttle protein (NSP) of Indian cassava mosaic virus using green fluorescent protein as reporter.

Indian cassava mosaic virus (ICMV), responsible for the cassava mosaic disease in India, harbours two circular genomic components, DNA-A and DNA-B; the former being responsible for the encapsidation and replication and the latter for intra- and inter-cellular movement of the viral DNA. Two proteins, encoded by DNA-B, the movement protein (MP) and the nuclear shuttle protein (NSP), act in concert on the newly replicated viral DNA to move it from the nucleus to the cell periphery. To map the functional domains of NSP, the intra-cellular localization of its full-length protein and deletion derivatives was studied in the epidermal cells of detached leaves of the laboratory host plant, Nicotiana benthamiana, where the target proteins were transiently expressed as GFP fusions. This analysis revealed domains for nuclear localization at the N-terminus, as well as for localization towards the cell periphery both at the C-terminus and center of the NSP.

Chronic kidney disease, physical activity and cognitive function in older adults-results from the National Health and Nutrition Examination Survey (2011-2014).

BACKGROUND: Cognitive impairment is common among persons with chronic kidney disease (CKD), due in part to reduced kidney function. Given that physical activity (PA) is known to mitigate cognitive decline, we examined whether associations between CKD stage and global/domain-specific cognitive function differ by PA. METHODS: We leveraged 3223 participants (≥60 years of age) enrolled in National Health and Nutrition Examination Survey (NHANES, 2011-2014), with at least one measure of objective cognitive function [immediate recall (CERAD-WL), delayed recall (CERAD-DR), verbal fluency (AF), executive function/processing speed (DSST), global (average of four tests) or self-perceived memory decline (SCD)]. We quantified the association between CKD stage {no CKD: estimated glomerular filtration rate [eGFR] ≥60 mL/min/1.73 m2 and albuminuria [albumin:creatinine ratio (ACR)] <30 mg/g; stages G1-G3: eGFR ≥60 mL/min/1.73 m2 and ACR ≥30 mg/g or eGFR 30-59 mL/min/1.73 m2; stages G4 and G5: eGFR <30 mL/min/1.73 m2} and cognitive function using linear regression (objective measures) and logistic regression (SCD), accounting for sampling weights for nationally representative estimates. We tested whether associations differed by PA [Global Physical Activity Questionnaire, high PA ≥600 metabolic equivalent of task (MET) · min/week versus low PA <600 MET · min/week] using a Wald test. RESULTS: Among NHANES participants, 34.9% had CKD stages G1-G3, 2.6% had stages G4 and G5 and 50.7% had low PA. CKD stages G4 and G5 were associated with lower global cognitive function {difference = -0.38 standard deviation [SD] [95% confidence interval (CI) -0.62 to -0.15]}. This association differed by PA (Pinteraction = 0.01). Specifically, among participants with low PA, those with CKD stages G4 and G5 had lower global cognitive function [difference = -0.57 SD (95% CI -0.82 to -0.31)] compared with those without CKD. Among those with high PA, no difference was found [difference = 0.10 SD (95% CI -0.29-0.49)]. Similarly, the CKD stage was only associated with immediate recall, verbal fluency, executive function and processing speed among those with low PA; no associations were observed for delayed recall or self-perceived memory decline. CONCLUSIONS: CKD is associated with lower objective cognitive function among those with low but not high PA. Clinicians should consider screening older patients with CKD who have low PA for cognitive impairment and encourage them to meet PA guidelines.

Acute kidney injury is more common in men than women after accounting for socioeconomic status, ethnicity, alcohol intake and smoking history.

BACKGROUND: The association of several comorbidities, including diabetes mellitus, hypertension, cardiovascular disease, heart failure and chronic kidney or liver disease, with acute kidney injury (AKI) is well established. Evidence on the effect of sex and socioeconomic factors are scarce. This study was designed to examine the association of sex and socioeconomic factors with AKI and AKI-related mortality and further to evaluate the additional relationship with other possible risk factors for AKI occurrence. METHODS: We included 3534 patients (1878 males with mean age 61.1 ± 17.7 and 1656 females 1656 with mean age 60.3 ± 20.0 years) admitted to Queen Elizabeth or Heartlands Hospitals, Birmingham, between October 2013 and January 2016. Patients were prospectively followed-up for a median 47.70 [IQR, 18.20] months. Study-endpoints were incidence of AKI, based on KDIGO-AKI Guidelines, and all-cause mortality. Data acquisition was automated, and information on mortality was collected from the Hospital Episode Statistics and Office of National Statistics. Socioeconomic status was evaluated with the Index of Multiple Deprivation (IMD). RESULTS: Incidence of AKI was higher in men compared to women (11.3% vs 7.1%; P < 0.001). Model regression analysis revealed significant association of male sex with higher AKI risk (OR, 1.659; 95% CI, 1.311-2.099; P < 0.001); this association remained significant after adjustment for age, eGFR, IMD, smoking, alcohol consumption, ethnicity, existing comorbidities and treatment (OR, 1.599; 95% CI, 1.215-2.103; P = 0.001). All-cause mortality was higher in patients with compared to those without AKI. Males with AKI had higher mortality rates in the first 6-month and 1-year periods after the index AKI event. The association of male sex with mortality was independent of socioeconomic factors but was not statistically significant after adjustment for existing comorbidities. CONCLUSIONS: Men are at higher risk of AKI and this association is independent from existing risk factors for AKI. The association between male sex and AKI-related mortality was not independent from existing comorbidities. A better understanding of factors associated with AKI may help accurately identify high-risk patients.

Kidney transplant dysfunction in a patient with COVID - 19 infection: role of concurrent Sars-Cov 2 nephropathy, chronic rejection and vitamin C-mediated hyperoxalosis: case report.

BACKGROUND: COVID-19 infection in kidney transplant recipients often lead to allograft dysfunction. The allograft injury has various histopathological manifestations. Our case illustrates the unusual combination of allograft rejection, acute kidney injury secondary to oxalate nephropathy and SARS CoV-2 nephropathy as the cause of irreversible allograft failure. CASE PRESENTATION: A 56 year old renal allograft recipient presented with a history of fever and diarrhoea for the preceding 4 weeks, tested positive for Sars-CoV2 on nasal swab and was found to have severe allograft dysfunction, necessitating haemodialysis. He subsequently underwent an allograft biopsy, which demonstrated antibody mediated rejection along with the presence of extensive oxalate deposition in the tubules. Ultrastructural examination demonstrated spherical spiked particles in the glomerular capillary endothelium and the presence of tubulo-reticular inclusions suggestive of an active COVID-19 infection within the kidney. The intra-tubular oxalate deposition was considered to be the result of high dose, supplemental Vitamin C used as an immune booster in many patients with COVID - 19 infection in India. CONCLUSIONS: This case highlights the complex pathology that may be seen in following COVID-19 disease and the need for kidney biopsies in these patients to better understand the aetiology of disease.

Association between non-malignant monoclonal gammopathy and adverse outcomes in chronic kidney disease: A cohort study.

BACKGROUND: In studies including the general population, the presence of non-malignant monoclonal gammopathy (MG) can be causally associated with kidney damage and shorter survival. We assessed whether the presence of an MG is associated with a higher risk of kidney failure or death in individuals with chronic kidney disease (CKD). METHODS AND FINDINGS: Data were used from 3 prospective cohorts of individuals with CKD (not on dialysis or with a kidney transplant): (1) Renal Impairment in Secondary Care (RIISC, Queen Elizabeth Hospital and Heartlands Hospital, Birmingham, UK, N = 878), (2) Salford Kidney Study (SKS, Salford Royal Hospital, Salford, UK, N = 861), and (3) Renal Risk in Derby (RRID, Derby, UK, N = 1,739). Participants were excluded if they had multiple myeloma or any other B cell lymphoproliferative disorder with end-organ damage. Median age was 71.0 years, 50.6% were male, median estimated glomerular filtration rate was 42.3 ml/min/1.73 m2, and median urine albumin-to-creatinine ratio was 3.4 mg/mmol. All non-malignant MG was identified in the baseline serum of participants of RIISC. Further, light chain MG (LC-MG) was identified and studied in participants of RIISC, SKS, and RRID. Participants were followed up for kidney failure (defined as the initiation of dialysis or kidney transplantation) and death. Associations with the risk of kidney failure were estimated by competing-risks regression (handling death as a competing risk), and associations with death were estimated by Cox proportional hazards regression. In total, 102 (11.6%) of the 878 RIISC participants had an MG. During a median follow-up time of 74.0 months, there were 327 kidney failure events and 202 deaths. The presence of MG was not associated with risk of kidney failure (univariable subhazard ratio [SHR] 0.97 [95% CI 0.68 to 1.38], P = 0.85; multivariable SHR 1.16 [95% CI 0.80 to 1.69], P = 0.43), and although there was a higher risk of death in univariable analysis (hazard ratio [HR] 2.13 [95% CI 1.49 to 3.02], P < 0.001), this was not significant in multivariable analysis (HR 1.37 [95% CI 0.93 to 2.00], P = 0.11). Fifty-five (1.6%) of the 3,478 participants from all 3 studies had LC-MG. During a median follow-up time of 62.5 months, 564 of the 3,478 participants progressed to kidney failure, and 803 died. LC-MG was not associated with risk of kidney failure (univariable SHR 1.07 [95% CI 0.58 to 1.96], P = 0.82; multivariable SHR 1.42 [95% CI 0.78 to 2.57], P = 0.26). There was a higher risk of death in those with LC-MG in the univariable model (HR 2.51 [95% CI 1.59 to 3.96], P < 0.001), but not in the multivariable model (HR 1.49 [95% CI 0.93 to 2.39], P = 0.10). An important limitation of this work was that only LC-MG, rather than any MG, could be identified in participants from SKS and RRID. CONCLUSIONS: The prevalence of MG was higher in this CKD cohort than that reported in the general population. However, the presence of an MG was not independently associated with a significantly higher risk of kidney failure or, unlike in the general population, risk of death.

Artificially induced phased siRNAs promote virus resistance in transgenic plants.

We previously developed transgenic tobacco plants that were resistant to two geminiviruses. We generated resistance using RNAi constructs that produced trans-acting siRNA (tasiRNA) like secondary siRNAs known as phased siRNA (phasiRNA) that targeted several regions of Tomato Leaf Curl New Delhi Virus (ToLCNDV) and Tomato Leaf Curl Gujarat Virus (ToLCGV) transcripts encoding the RNA silencing suppressor proteins AC2 and AC4. Here, we performed degradome analysis to determine the precise cleavage sites of RNA-RNA interaction between phasiRNA and viral transcripts. We then applied our RNAi technology in tomato, which is the natural host for ToLCNDV and ToLCGV. The relative ease of developing and using phasiRNA constructs represents a significant technical advance in imparting virus resistance in crops and/or important model systems.

Associations between Hemodialysis Facility Practices to Manage Fluid Volume and Intradialytic Hypotension and Patient Outcomes.

BACKGROUND AND OBJECTIVES: Fluid overload and intradialytic hypotension are associated with cardiovascular events and mortality in patients on hemodialysis. We investigated associations between hemodialysis facility practices related to fluid volume and intradialytic hypotension and patient outcomes. DESIGN, SETTING, PARTICIPANTS, & MEASUREMENTS: Data were analyzed from 10,250 patients in 273 facilities across 12 countries, from phase 4 of the Dialysis Outcomes and Practice Patterns Study (DOPPS; 2009-2012). Cox regression models (shared frailty) were used to estimate associations between facility practices reported by medical directors in response to the DOPPS Medical Directors Survey and all-cause and cardiovascular mortality and hospitalization, and cardiovascular events, adjusting for country, age, sex, dialysis vintage, predialysis systolic BP, cardiovascular comorbidities, diabetes, body mass index, smoking, residual kidney function, dialysis adequacy, and vascular access type. RESULTS: Of ten facility practices tested (chosen a priori), having a protocol that specifies how often to assess dry weight in most patients was associated with lower all-cause (hazard ratio [HR], 0.78; 99% confidence interval [99% CI], 0.64 to 0.94) and cardiovascular mortality (HR, 0.72; 99% CI, 0.55 to 0.95). Routine orthostatic BP measurement to assess dry weight was associated with lower all-cause hospitalization (HR, 0.86; 99% CI, 0.77 to 0.97) and cardiovascular events (HR, 0.85; 99% CI, 0.73 to 0.98). Routine use of lower dialysate temperature to limit or prevent intradialytic hypotension was associated with lower cardiovascular mortality (HR, 0.76; 99% CI, 0.58 to 0.98). Routine use of an online volume indicator to assess dry weight was associated with higher all-cause hospitalization (HR, 1.19; 99% CI, 1.02 to 1.38). Routine use of sodium modeling/profiling to limit or prevent intradialytic hypotension was associated with higher all-cause mortality (HR, 1.36; 99% CI, 1.14 to 1.63), cardiovascular mortality (HR, 1.34; 99% CI, 1.04 to 1.73), and cardiovascular events (HR, 1.21; 99% CI, 1.03 to 1.43). CONCLUSIONS: Hemodialysis facility practices relating to the management of fluid volume and intradialytic hypotension are associated with patient outcomes.

Rationale and design of BISTRO: a randomized controlled trial to determine whether bioimpedance spectroscopy-guided fluid management maintains residual kidney function in incident haemodialysis patients.

BACKGROUND: Preserved residual kidney function (RKF) and normal fluid status are associated with better patient outcomes in incident haemodialysis patients. The objective of this trial is to determine whether using bioimpedance technology in prescribing the optimal post-dialysis weight can reduce the rate of decline of RKF and potentially improve patient outcomes. METHODS/DESIGN: 516 pateints commencing haemodialysis, aged >18 with RKF of > 3 ml/min/1.73 m2 or a urine volume >500 ml per day or per the shorter inter-dialytic period will be consented and enrolled into a pragmatic, open-label, randomized controlled trial. The intervention is incorporation of bioimpedance spectroscopy (BI) determination of normally hydrated weight to set a post-dialysis target weight that limits volume depletion, compared to current standard practice. Clinicians and participants will be blinded to BI measures in the control group and a standardized record capturing management of fluid status will be used in all participants. Primary outcome is preservation of residual kidney function assessed as time to anuria (≤100 ml/day or ≤200 ml urine volume in the short inter-dialytic period). A sample size of 516 was based upon a cumulative incidence of 30% anuria in the control group and 20% in the treatment group and 11% competing risks (death, transplantation) over 10 months, with up to 2 years follow-up. Secondary outcomes include rate of decline in small solute clearance, significant adverse events, hospitalization, loss of vascular access, cardiovascular events and interventions, dialysis efficacy and safety, dialysis-related symptoms and quality of life. Economic evaluation will be carried out to determine the cost-effectiveness of the intervention. Analyses will be adjusted for patient characteristics and dialysis unit practice patterns relevant to fluid management. DISCUSSION: This trial will establish the added value of undertaking BI measures to support clinical management of fluid status and establish the relationship between fluid status and preservation of residual kidney function in incident haemodialysis patients. TRIAL REGISTRATION: ISCCTN Number: 11342007 , completed 26/04/2016; NIHR Portfolio number: CPMS31766; Sponsor: Keele University.

Identification of amino acid residues of the coat protein of Sri Lankan cassava mosaic virus affecting symptom production and viral titer in Nicotiana benthamiana.

Sri Lankan cassava mosaic virus (SLCMV) is bipartite begomovirus infecting cassava in India and Sri Lanka. Interestingly, the DNA-A component of the SLCMV alone is able to infect Nicotiana benthamiana causing symptoms of upward leaf rolling and stunting. One of the differences between monopartite and bipartite begomoviruses is the requirement of Coat Protein (CP) for infectivity; CP being essential for the former, but dispensable in the latter. This investigation was aimed to determine the importance of CP in the infectivity of the bipartite SLCMV, behaving as a monopartite virus in N. benthamiana. We tested CP-null mutants, single amino acid replacement mutants and double, triple and quadruple combinations of the above in SLCMV DNA-A, for infectivity, symptom development and viral DNA accumulation in N. benthamiana. While CP-null mutants were non-infectious, a majority of the single amino acid replacement mutants and their combinations retained infectivity, some with attenuated symptoms and reduced viral titers. Some of the combined mutations restored the attenuated symptoms to wild type levels. Some of the mutations were predicted to cause changes in the secondary structure of the CP, which roughly correlated with the attenuation of symptoms and the reduction in viral titers.

Predicting non-diabetic renal disease in type 2 diabetic adults: The value of glycated hemoglobin.

AIMS: The indications for renal biopsy in type 2 diabetes mellitus (T2D) are not well established. We investigated the prevalence, spectrum, and predictors of biopsy-proven non-diabetic renal disease (NDRD) in T2D. METHODS: An observational, single-center, retrospective study of T2D adults who underwent renal biopsies (N = 51) over 10 years for nephrotic-range proteinuria, microscopic hematuria, or rapidly declining renal function. RESULTS: Thirty-five (68.6%) biopsies were diagnostic of NDRD, and 16 (31.4%) revealed isolated diabetic nephropathy. The most common NDRDs were interstitial nephritis (20%), progressive crescentic glomerulonephritis (14%), membranous nephropathy (11%), and focal segmental glomerulosclerosis (11%). The odds for NDRD declined by 97% in the presence of diabetic retinopathy (P < 0.001). The deterioration of HbA1c during the year before biopsy predicted NDRD even after adjusting for diabetic retinopathy (OR, 7.65; 95% CI, 1.36-123.04; P = 0.003). A model based on the interaction between the HbA1c values 12 months before biopsy and the absolute change in these values during the preceding year predicted NDRD with 73.7% sensitivity and 75% specificity (AUC, 0.77; 95% CI, 0.59-0.94). CONCLUSIONS: This study demonstrated a considerably high prevalence of NDRD in T2D adults undergoing renal biopsy. The absence of diabetic retinopathy, lower HbA1c values 12 months before biopsy and greater deterioration in HbA1c prior to biopsy predicted NDRD in T2D. Further studies are needed to validate the findings.

Development of plants resistant to tomato geminiviruses using artificial trans-acting small interfering RNA.

RNA interference (RNAi), a conserved RNA-mediated gene regulatory mechanism in eukaryotes, plays an important role in plant growth and development, and as an antiviral defence system in plants. As a counter-strategy, plant viruses encode RNAi suppressors to suppress the RNAi pathways and consequently down-regulate plant defence. In geminiviruses, the proteins AC2, AC4 and AV2 are known to act as RNAi suppressors. In this study, we have designed a gene silencing vector using the features of trans-acting small interfering RNA (tasiRNA), which is simple and can be used to target multiple genes at a time employing a single-step cloning procedure. This vector was used to target two RNAi suppressor proteins (AC2 and AC4) of the geminivirus, Tomato leaf curl New Delhi virus (ToLCNDV). The vector containing fragments of ToLCNDV AC2 and AC4 genes, on agro-infiltration, produced copious quantities of AC2 and AC4 specific siRNA in both tobacco and tomato plants. On challenge inoculation of the agro-infiltrated plants with ToLCNDV, most plants showed an absence of symptoms and low accumulation of viral DNA. Transgenic tobacco plants were raised using the AC2 and AC4 tasiRNA-generating constructs, and T1 plants, obtained from the primary transgenic plants, were tested for resistance separately against ToLCNDV and Tomato leaf curl Gujarat virus. Most plants showed an absence of symptoms and low accumulation of the corresponding viruses, the resistance being generally proportional to the amounts of siRNA produced against AC2 and AC4 genes. This is the first report of the use of artificial tasiRNA to generate resistance against an important plant virus.

The effect of biliopancreatic diversion surgery on renal function--a retrospective study.

BACKGROUND: The prevalences of obesity and chronic kidney disease (CKD) have increased simultaneously. Should a pathophysiological relationship exist between the two conditions, bariatric surgery and associated weight loss could be an important intervention in extremely obese individuals to slow the progression of CKD. METHODS: We conducted a retrospective analysis of 25 patients who had undergone biliopancreatic diversion (BPD) surgery for extreme obesity (body mass index >40 kg/m(2)), with mean follow-up of 4 years. We assessed pre- and post-surgery renal function, body weight and blood pressure (BP) obtained from electronic hospital and primary care records. RESULTS: There was a significant reduction in mean body weight at 4 years by 50.3 kg (SD = 20.65). The creatinine and estimated glomerular filtration rate (eGFR) also improved significantly: serum creatinine reduced by 16.2 µmol/l (SD = 19.57) while the eGFR improved by 10.6 ml/min/m(2) (SD = 15.45). The greatest improvement in eGFR was in the group (n = 7) with eGFR ≤60 ml/min/m(2). A subset of patients (n = 11) had evaluable BP readings and had a reduction in BP of 17/10 mmHg (SD = 33/12). CONCLUSIONS: This retrospective study demonstrates a clinically significant improvement in renal function following BPD. Several mechanisms including weight loss could account for the positive impact on renal function. The physiology underlying this improvement requires further study.

Agroinfection of cloned Sri Lankan cassava mosaic virus DNA to Arabidopsis thaliana, Nicotiana tabacum and cassava.

Sri Lankan cassava mosaic virus (SLCMV) is a bipartite begomovirus infecting cassava in India and Sri Lanka. We have used Agrobacterium-mediated inoculation (agroinoculation) of cloned SLCMV DNA to inoculate additional hosts, Nicotiana tabacum and Arabidopsis. Although SLCMV infection in these hosts caused stunting, leaf deformation and developmental abnormalities, accumulation levels of viral DNA in the infected plants suggested that this virus was poorly adapted to them. In the natural host, cassava, agroinoculation produced infection at a low frequency. The monopartite nature of SLCMV, reported earlier in N. benthamiana, was maintained in the new hosts as well as in cassava.

Virus-induced gene silencing for functional analysis of selected genes.

Virus-induced gene silencing (VIGS) is a technology that exploits an RNA-mediated antiviral defense mechanism and has been shown to be of great potential in plant reverse genetics. Circumvention of plant transformation, methodological simplicity, robustness, and speedy results makes VIGS an attractive alternative instrument in functional genomics, even in a high throughput fashion. The system is well established in Nicotiana benthamiana, and efforts are being made to improve VIGS in other species, including monocots. Here, we discuss the issues specific to the application of VIGS technology to determine gene function, which has revealed the roles of a variety of genes in disease resistance, abiotic stress, cellular signaling and secondary metabolite biosynthesis.

Downstream promoter sequence of an Indian isolate of Rice tungro bacilliform virus alters tissue-specific expression in host rice and acts differentially in heterologous system.

An Indian isolate of Rice tungro bacilliform virus from West Bengal (RTBV-WB) showed significant nucleotide differences in its putative promoter region when compared with a previously characterized isolate from Philippines. The transcription start site of RTBV-WB was mapped followed by assessing the activity and tissue-specificity of the full-length (FL) promoter (-231 to +645) and several of its upstream and downstream deletions by studying the expression of beta-Glucuronidase (GUS) reporter gene in transgenic rice (Oryza sativa L. subsp. indica) plants at various stages of development. In addition to the expected vascular-specific expression pattern, studied by histochemical staining, GUS enzymatic assay and northern and RT-PCR analysis, two novel patterns were revealed in some of the downstream deleted versions; a non-expressing type, representing no expression at any stage in any tissue and constitutive type, representing constitutive expression at all stages in most tissues. This indicated the presence of previously unreported positive and negative cis-regulatory elements in the downstream region. The negative element and a putative enhancer region in the upstream region specifically bound to rice nuclear proteins in vitro. The FL and its deletion derivatives were also active in heterologous systems like tobacco (Nicotiana tabacum) and wheat (Triticum durum). Expression patterns in tobacco were different from those observed in rice suggesting the importance of upstream elements in those systems and host-specific regulation of the promoter in diverse organisms. Thus, the RTBV-WB FL promoter and its derivatives contain an array of cis-elements, which control constitutive or tissue- and development-specific gene expression in a combinatorial fashion.

Deletion and recombination events between the DNA-A and DNA-B components of Indian cassava-infecting geminiviruses generate defective molecules in Nicotiana benthamiana.

Cloned DNA-B components, belonging to the bipartite begomoviruses Indian cassava mosaic virus (ICMV) and Sri Lankan cassava mosaic virus (SLCMV), family Geminiviridae, when co-inoculated along with previously cloned DNA-A components of the respective viruses onto the experimental host Nicotiana benthamiana, generated defective DNAs (def-DNA) ranging in size from 549 to 1555 nucleotides. All the cloned def-DNAs contained the common region (CR) as well as portions of either DNA-A or DNA-B and, in a few cases, both DNA-A and DNA-B, representing recombinant products, the junction points of which correspond to repeats of 2-11 bases found in the parental molecules. The DNA-B-derived def-DNAs were, in some cases, associated with a decrease in levels of DNA-B, with a concomitant change in the symptoms from downward leaf curling in the older leaves to upward leaf-rolling in newly emerging leaves, more typical of monopartite begomoviruses.