Characterization of Type1 Lipid Transfer Protein from Citrus sinensis: Unraveling its potential as an antimicrobial and insecticidal agent.

Lipid Transfer Protein1 (LTP1) is a cationic, multifaceted protein belonging to the pathogenesis-related protein (PR14) family. Despite being involved in diverse physiological processes and defense mechanisms, the precise in-vivo role of LTP1 remains undiscovered. This work presents the characterization of recombinant Citrus sinensis LTP1 (CsLTP1) along with lipid binding studies through in-silico and in-vitro approaches. CsLTP1 demonstrated great thermal and pH stability with a huge biotechnological potential. It showed in-vitro binding capacity with jasmonic acid and lipids involved in regulating plant immune responses. Gene expression profiling indicated a significant upregulation of CsLTP1 in Candidatus-infected Citrus plants. CsLTP1 disrupted the cell membrane integrity of various pathogens, making it a potent antimicrobial agent. Further, in-vivo antimicrobial and insecticidal properties of CsLTP1 have been explored. The impact of exogenous CsLTP1 treatment on rice crop metabolism for managing blight disease has been studied using GC-MS. CsLTP1 triggered crucial metabolic pathways in rice plants while controlling the blight disease. CsLTP1 effectively inhibited Helicoverpa armigera larvae by impeding mid-gut α-amylase activity and obstructing its developmental stages. This study highlights the pivotal role of CsLTP1 in plant defense by offering insights for developing multi-target therapeutic agent or disease-resistant varieties to comprehensively tackle the challenges towards crop protection.

Application of nanoparticles for management of plant viral pathogen: Current status and future prospects.

Plant viruses are responsible for nearly 47 % of all crop losses brought by plant diseases, which have a considerable negative impact on agricultural output. Nanoparticles have the potential to greatly raise agricultural output due to their wonderful applications in the fields of highly sensitive biomolecular detection, disease diagnostics, antimicrobials, and therapeutic compounds. The application of nanotechnology in plant virology is known as nanophytovirology, and it involves biostimulation, drug transport, genetic manipulation, therapeutic agents, and induction of plant defenses. The inactivation and denaturation of capsid protein, nucleic acids (RNA or DNA), and other protein constituents are involved in the underlying mechanism. To determine the precise mechanism by which nanoparticles affect viral mobility, reproduction, encapsidation, and transmission, more research is however required. Nanoparticles can be used to precisely detect plant viruses using nanobiosensors or as biostimulants. The varieties of nanoparticles employed in plant virus control and their methods of virus suppression are highlighted in this review.

Comparative Analysis of Inhibitor Binding to Peroxiredoxins from Candidatus Liberibacter asiaticus and Its Host Citrus sinensis.

The peroxiredoxins (Prxs), potential drug targets, constitute an important class of antioxidant enzymes present in both pathogen and their host. The comparative binding potential of inhibitors to Prxs from pathogen and host could be an important step in drug development against pathogens. Huanglongbing (HLB) is a most devastating disease of citrus caused by Candidatus Liberibacter asiaticus (CLa). In this study, the binding of conoidin-A (conoidin) and celastrol inhibitor molecules to peroxiredoxin of bacterioferritin comigratory protein family from CLa (CLaBCP) and its host plant peroxiredoxin from Citrus sinensis (CsPrx) was assessed. The CLaBCP has a lower specific activity than CsPrx and is efficiently inhibited by conoidin and celastrol molecules. The biophysical studies showed conformational changes and significant thermal stability of CLaBCP in the presence of inhibitor molecules as compared to CsPrx. The surface plasmon resonance (SPR) studies revealed that the conoidin and celastrol inhibitor molecules have a strong binding affinity (KD) with CLaBCP at 33.0 µM, and 18.5 µM as compared to CsPrx at 52.0 µM and 61.6 µM, respectively. The docked complexes of inhibitor molecules showed more structural stability of CLaBCP as compared to CsPrx during the run of molecular dynamics-based simulations for 100 ns. The present study suggests that the conoidin and celastrol molecules can be exploited as potential inhibitor molecules against the CLa to manage the HLB disease.

Biochemical characterization and structure-based in silico screening of potent inhibitor molecules against the 1 cys peroxiredoxin of bacterioferritin comigratory protein family from Candidatus Liberibacter asiaticus.

Bacterioferritin comigratory protein family 1 Cys peroxiredoxin from Candidatus Liberibacter asiaticus (CLaBCP) is an important antioxidant defense protein that participates in the reduction of ROS, free radicals, and peroxides. In the present study, we report the biochemical studies and in silico screening of potent antibacterial molecules against CLaBCP. The CLaBCP showed enzymatic activity with the Km value 54.43, 94.34, 120.6 µM, and Vmax of 59.37, 69.37, 70.0 µM min-1 for H2O2, TBHP, CHP respectively. The residual peroxidase activity of CLaBCP was analyzed at different ranges of pH and temperatures. The CLaBCP showed structural changes and unfolding in the presence of its substrates and guanidinium chloride by CD and fluorescence. The structure-based drug design method was employed to screen and identify the more efficient molecule against CLaBCP. The validated CLaBCP model was used for the virtual screening of potent antibacterial molecules. The docking was performed at CLaBCP active site to evaluate the binding energy of the top five molecules (LAS 34150849, BDE 33184869, LAS 51497689, BDE 33672484, and LAS 34150966). All identified molecule has a higher binding affinity than adenanthin analyzed by molecular docking. Molecular dynamics studies such as RMSD, Rg, SASA, and PCA results showed that the CLaBCP inhibitor(s) complex is more stable than the CLaBCP-adenanthin complex. MMPBSA results suggested that the identified molecule could form a lower energy CLaBCP-inhibiter(s) complex than the CLaBCP-adenanthin complex. The screened molecules may pave the route for the development of potent antibacterial molecules against CLa.Communicated by Ramaswamy H. Sarma.

Symptoms and Prevalence of Constipation among Adult Population of Bangladesh.

Background: Constipation is one of the most common gastrointestinal disorders. The prevalence of constipation is rapidly increasing globally. It has adverse effects on the patient's quality of life including productivity and results in a high financial hardship on the healthcare system. The aim of the study was to estimate the symptoms and prevalence of constipation among the adult population of Bangladesh. Materials and methods: It was a cross-sectional observational study based on a structured questionnaire and a checklist. In this study, three criteria were used for the diagnosis of chronic constipation (self-reported perception, Rome III criteria, and Bristol's criteria). The study was conducted among 1,550 population between July 2019 and December 2019. Result: The study population consisted of 1,550 respondents, among them 41.61% male and 58.39% female, and the mean age was 32.71 ± 9.72 years. In the study, 12.2% of the population was categorized to have constipation according to self-reported perception, 11.2% according to Rome III, and 10.3% reported to have been suffering from constipation according to Bristol chart.Female gender tends to have a greater prevalence than male. In multivariate analysis for constipation, betel nut chewer, alcohol consumer, diabetes mellitus, hypertension, GI surgery, and bronchial asthma were significantly (p < 0.001) associated with constipation. According to Bristol's criteria, the most common stool form was Type III (sausage-shaped with cracked surface) among the Bangladeshi population in this study. Conclusion: Chronic constipation is a common problem worldwide. The findings of this study suggest that there is a high prevalence of constipation among the general population of Bangladesh. Decreasing modifiable risk factors of constipation can reduce its prevalence and burden of the disease. Bangladesh is markedly deficient in literature citing constipation prevalence and determinants. These findings may commence a call for setting priority as one of the major public health problems and demanding attention for both at the clinical and community levels. How to cite this article: Ghosh DK, Sarkar DK, Nath M, et al. Symptoms and Prevalence of Constipation among Adult Population of Bangladesh. Euroasian J Hepato-Gastroenterol 2023;13(2):45-49.

Characterization of a cytoplasmic 2-Cys peroxiredoxin from Citrus sinensis and its potential role in protection from oxidative damage and wound healing.

In present work, the recombinant cytoplasmic 2-Cys peroxiredoxin from Citrus sinensis (CsPrx) was purified and characterized. The peroxidase activity was examined with different substrates using DTT, a non-physiological electron donor. The conformational studies, in oxidized and reduced states, were performed using circular dichroism (CD) and fluorescence measurement. The CD analysis showed higher α-helical content for reduced state of the protein. The thermal stability studies of CsPrx by Differential Scanning Calorimetry (DSC) showed that oxidized state is more stable as compared to the reduced state of CsPrx. In vitro studies showed that the CsPrx provides a protective shield against ROS and free radicals that participate in the degradation of plasmid DNA. The pre-treatment of 10 µM CsPrx provide almost 100% protection against peroxide-mediated cell killing in the Vero cells. CsPrx showed significant cell proliferation and wound healing properties. The superior morphology of viable cells and wound closure was found at 20 µM CsPrx treated for 12 h. The results demonstrated that CsPrx is a multifaceted protein with a significant role in cell proliferation, wound healing and protection against hydrogen peroxide-induced cellular damage. This could be the first report of a plant peroxiredoxin being characterized for biomedical applications.

A Comprehensive Analysis of Citrus Tristeza Variants of Bhutan and Across the World.

Mandarin orange is economically one of the most important fruit crops in Bhutan. However, in recent years, orange productivity has dropped due to severe infection of citrus tristeza virus (CTV) associated with the gradual decline of citrus orchards. Although the disease incidence has been reported, very limited information is available on genetic variability among the Bhutanese CTV variants. This study used reverse transcription PCR (RT-PCR) to detect CTV in collected field samples and recorded disease incidence up to 71.11% in Bhutan's prominent citrus-growing regions. To elucidate the extent of genetic variabilities among the Bhutanese CTV variants, we targeted four independent genomic regions (5'ORF1a, p25, p23, and p18) and analyzed a total of 64 collected isolates. These genomic regions were amplified and sequenced for further comparative bioinformatics analysis. Comprehensive phylogenetic reconstructions of the GenBank deposited sequences, including the corresponding genomic locations from 53 whole-genome sequences, revealed unexpected and rich diversity among Bhutanese CTV variants. A resistant-breaking (RB) variant was also identified for the first time from the Asian subcontinent. Our analyses unambiguously identified five (T36, T3, T68, VT, and HA16-5) major, well-recognized CTV strains. Bhutanese CTV variants form two additional newly identified distinct clades with higher confidence, B1 and B2, named after Bhutan. The origin of each of these nine clades can be traced back to their root in the north-eastern region of India and Bhutan. Together, our study established a definitive framework for categorizing global CTV variants into their distinctive clades and provided novel insights into multiple genomic region-based genetic diversity assessments, including their pathogenicity status.

In-silico screening and identification of potential inhibitors against 2Cys peroxiredoxin of Candidatus Liberibacter asiaticus.

Huanglongbing (HLB) is a worldwide citrus plant disease-related to non-culturable and fastidious α-proteobacteria Candidatus Liberibacter asiaticus (CLas). In CLas, Peroxiredoxin (Prx) plays a major role in the reduction of the level of reactive species such as reactive oxygen species (ROS), free radicals and peroxides, etc. Here, we have used structure-based drug designing approach was used to screen and identify the potent molecules against 2Cys Prx. The virtual screening of fragments library was performed against the three-dimensional validated model of Prx. To evaluate the binding affinity, the top four molecules (N-Boc-2-amino isobutyric acid (B2AI), BOC-L-Valine (BLV), 1-(boc-amino) cyclobutane carboxylic acid (1BAC), and N-Benzoyl-DL-alanine (BDLA)) were docked at the active site of Prx. The molecular docking results revealed that all the identified molecules had a higher binding affinity than Tert butyl hydroperoxide (TBHP), a substrate of Prx. Molecular dynamics analysis such as RMSD, Rg, SASA, hydrogen bonds, and PCA results indicated that Prx-inhibitor(s) complexes had lesser fluctuations and were more stable and compact than Prx-TBHP complex. MMPBSA results confirmed that the identified compounds could bind at the active site of Prx to form a lower energy Prx-inhibitor(s) complex than Prx-TBHP complex. The identified potent molecules may pave the path for the development of antimicrobial agents against CLA.Communicated by Ramaswamy H. Sarma.

Development of a real-time RT-PCR method for the detection of Citrus tristeza virus (CTV) and its implication in studying virus distribution in planta.

Tristeza is an economically important disease of the citrus caused by Citrus tristeza virus (CTV) of genus Closterovirus and family Closteroviridae. The disease has caused tremendous losses to citrus industry worldwide by killing millions of trees, reducing the productivity and total production. Enormous efforts have been made in many countries to prevent the viral spread and the losses caused by the disease. To understand the reason behind this scenario, studies on virus distribution and tropism in the citrus plants are needed. Different diagnostic methods are available for early CTV detection but none of them is employed for in planta virus distribution study. In this study, a TaqMan RT-PCR-based method to detect and quantify CTV in different tissues of infected Mosambi plants (Citrus sinensis) has been standardized. The assay was very sensitive with the pathogen detection limit of > 0.0595 fg of in vitro-transcribed CTV-RNA. The assay was implemented for virus distribution study and absolute CTV titer quantification in samples taken from Tristeza-infected trees. The highest virus load was observed in the midribs of the symptomatic leaf (4.1 × 107-1.4 × 108/100 mg) and the lowest in partial dead twigs (1 × 103-1.7 × 104/100 mg), and shoot tip (2.3 × 103-4.5 × 103/100 mg). Interestingly, during the peak summer months, the highest CTV load was observed in the feeder roots (3 × 107-1.1 × 108/100 mg) than in the midribs of symptomatic leaf. The viral titer was highest in symptomatic leaf midrib followed by asymptomatic leaf midrib, feeder roots, twig bark, symptomatic leaf lamella, and asymptomatic leaf lamella. Overall, high CTV titer was primarily observed in the phloem containing tissues and low CTV titer in the other tissues. The information would help in selecting tissues with higher virus titer in disease surveillance that have implication in Tristeza management in citrus.

Development of a SYBR Green-based RT-qPCR assay for the detection of Indian citrus ringspot virus.

The Indian citrus ringspot virus (ICRSV) that causes ringspot disease, especially to 'Kinnow mandarin' hampers the sustainability of crop production. Presently, the disease is not amenable for control through host resistance or the introduction of chemicals, hence raising virus-free plants is one of the most effective approaches to manage the disease. Consequently, it is necessary to develop rapid, sensitive, specific, and early diagnostic methods for disease control. In the present study, newly designed primers targeting a 164 bp region of the ICRSV coat protein gene were used to develop and optimize a SYBR Green-based quantitative reverse transcription polymerase chain reaction (RT-qPCR) assay, for the detection of ICRSV. The RT-qPCR assay was evaluated and confirmed using viral RNA extracted from ICRSV infected plants maintained in screen house as well as field samples. The standard curves displayed a dynamic linear range across eight log units of ICRSV-cRNA copy number ranging from 9.48.1 fmol (5.709 × 109) to 0.000948 amol (5.709 × 102), with detection limit of 5.709 × 102 copies per reaction using serial tenfold diluted in vitro transcribed viral cRNA. The developed RT-qPCR is very specific to ICRSV does not react to other citrus pathogens, and approximately 100-fold more sensitive than conventional RT-PCR. Thus, this assay will be useful in laboratories, KVKs, and nurseries for the citrus budwood certification program as well as in plant quarantine stations. To our knowledge, this is the first study of the successful detection of ICRSV by RT-qPCR.

Detection and Molecular Characterization of 'Candidatus Liberibacter asiaticus' and Citrus Tristeza Virus Associated with Citrus Decline in Bhutan.

Citrus, mainly mandarin (Citrus reticulata Blanco), is an economically important fruit crop in Bhutan. Despite having favorable agroclimatic conditions for citrus cultivation, the early decline of fruit-bearing orchards coupled with low crop productivity is a major concern among citrus growers. During a recent survey, an association of 'Candidatus Liberibacter asiaticus' (citrus greening) and citrus tristeza virus (CTV), either singly or as mixed infections in declined citrus trees, was recorded in all four major citrus-growing districts (Tsirang, Dagana, Zhemgang, and Sarpang). Using PCR-based diagnosis, a higher incidence of citrus greening (27.45%) and tristeza (70.58%) was observed in symptomatic field samples. Detection and characterization of 'Ca. L. asiaticus' was performed based on the 16S ribosomal DNA, prophage gene, 50S ribosomal rplA-rplJ gene, and tandem repeats of the CLIBASIA_01645 locus. Similarly, the coat protein, p23, and p18 genes were used as genetic markers for the detection and characterization of Bhutanese CTV. The 'Ca. L. asiaticus' isolates from Bhutan segregated into classes II and III based on the CLIBASIA_01645 locus, analogous to Indian isolates from the northeast region and Term-A based on the CLIBASIA_05610 locus. CTV isolates of Bhutan were observed as closely related to the VT strain, which is considered to be the most devastating. To the best of our knowledge, this is the first study on molecular characterization of 'Ca. L. asiaticus' and CTV isolates and their association with citrus decline in Bhutan.

A Rapid and Sensitive Reverse Transcription-Loop-Mediated Isothermal Amplification (RT-LAMP) Assay for the Detection of Indian Citrus Ringspot Virus.

Indian citrus ringspot virus (ICRSV) is a devastating pathogen that has a particularly deleterious effect on the 'Kinnow mandarin', a commercial citrus crop cultivated in the northwest of India. ICRSV belongs to the Mandarivirus genus within the family of Alphaflexiviridae and has a positive sense single-stranded RNA (ssRNA) genome consisting of six open reading frames (ORFs). Severe cases of ICRSV result in a significant reduction in both the yield and quality of crops. Consequently, there is an urgent need to develop methods to detect ICRSV in an accurate and timely manner. Current methods involve a two-step reverse transcription polymerase chain reaction (RT-PCR) that is time consuming. Here, we describe a novel, one-step reverse transcription loop-mediated isothermal amplification (RT-LAMP) method for the sensitive and rapid detection of ICRSV. To standardize the RT-LAMP assay, four different primers were designed and tested to target the coat protein gene of ICRSV. Amplification results were visualized by a color change after addition of SYBR Green I. The standardized RT-LAMP assay was highly specific and successfully detected all 35 ICRSV isolates tested from the Punjab and Haryana states of India. Furthermore, there was no cross-reaction with 17 isolates of five other citrus pathogens that are common in India. The ICRSV RT-LAMP assay developed in the present study is a simple, rapid, sensitive, specific technique. Moreover, the assay consists of only a single step and is more cost effective than existing methods. This is the first application of RT-LAMP for the detection of ICRSV. Our RT-LAMP assay is a powerful tool for the detection of ICRSV and will be particularly useful for large-scale indexing of field samples in diagnostic laboratories, in nurseries, and for quarantine applications.

Sub-TeV H

We suggest searching for the charged Higgs boson at the Large Hadron Collider (LHC) via cg→bH^{+}→btb[over ¯]. In the general two Higgs doublet model, extra top Yukawa couplings ρ_{tc} and ρ_{tt} can drive the disappearance of antimatter from the Universe, while c[over ¯]bH^{+} and t[over ¯]bH^{+} couple with strength ρ_{tc}V_{tb} and ρ_{tt}V_{tb}, respectively. For ρ_{tc},ρ_{tt}∼0.5, and m_{H^{+}}∼300-500 GeV, evidence could emerge from LHC run 2 data at hand and discovery by adding run 3 data in the near future.

Development of a reverse transcription recombinase polymerase based isothermal amplification coupled with lateral flow immunochromatographic assay (CTV-RT-RPA-LFICA) for rapid detection of Citrus tristeza virus.

Tristeza is a highly destructive disease of citrus caused by the phloem-limited, flexuous filamentous Citrus tristeza virus (CTV) in the genus Closterovirus and the family Closteroviridae. It has been a major constraint for higher productivity and has destroyed millions of citrus trees globally. CTV is graft transmissible and spread through use of virus infected nursery plants. Therefore, virus detection by using specific and reliable diagnostic tools is very important to mitigate disease outbreaks. Currently, the standard molecular techniques for CTV detection include RT-PCR and RT-qPCR. These diagnostic methods are highly sensitive but time consuming, labor intensive and require sophisticated expensive instruments, thus not suitable for point-of-care use. In the present study, we report the development of a rapid, sensitive, robust, reliable, and highly specific reverse transcription-RPA technique coupled with a lateral flow immunochromatographic assay (CTV-RT-RPA-LFICA). RT-RPA technique was standardized to amplify the coat protein gene of CTV (CTV-p25) and detect double labeled amplicons on a sandwich immunoassay by designing specific labeled primer pair and probe combinations. The optimally performing primer set (CTRPA-F1/CTRPA-R9-Btn) and the corresponding TwistAmp nfo probe (CTRPA-Probe) was optimized for temperature and reaction time using purified cDNA and viral RNA as template. The sensitivity of the developed assay was compared with other detection techniques using in vitro-transcribed RNA. The efficacy and specificity of the assay was evaluated using CTV positive controls, healthy samples, field grown citrus plants of unknown status, and other virus and bacterial pathogens that infect citrus plants. The RT-RPA-LFICA was able to detect ≤ 141 fg of RNA when cDNA used as a template. The assay detected ≤ 0.23 ng/µl of CTV RNA when directly used as template without cross-reactivity with other citrus pathogens. Best results were achieved at the isothermal temperature of 40 °C within 15-20 min. The study demonstrated that RT-RPA-LFICA has potential to become an improved detection technique for end users in bud-wood certification and quarantine programs and a promising platform for rapid point-of-care diagnostics for citrus farmers and small nurseries in low resource settings.

In-silico characterization and RNA-binding protein based polyclonal antibodies production for detection of citrus tristeza virus.

Citrus tristeza virus (CTV) is the etiologic agent of the destructive Tristeza disease, a massive impediment for the healthy citrus industry worldwide. Routine indexing of CTV is an essential component for disease surveys and citrus budwood certification for production of disease-free planting material. Therefore, the present study was carried out to develop an efficient serological assay for CTV detection based on the RNA binding protein (CTV-p23), which is translated from a subgenomic RNA (sgRNA) that accumulates at higher levels in CTV-infected plants. CTV-p23 gene was amplified, cloned and polyclonal antibodies were raised against recombinant CTV-p23 protein. The efficacy of the produced polyclonal antibodies was tested by Western blots and ELISA to develop a quick, sensitive and economically affordable CTV detection tool and was used for indexing of large number of plant samples. The evaluation results indicated that the developed CTV-p23 antibodies had an excellent diagnostic agreement with RT-PCR and would be effective for the detection of CTV in field samples. Furthermore, CTV-p23 gene specific primers designed in the present study were found 1000 times more sensitive than the reported coat protein (CTV-p25) gene specific primers for routine CTV diagnosis. In silico characterizations of CTV-p23 protein revealed the presence of key conserved amino acid residues that involved in the regulation of protein stability, suppressor activity and protein expression levels. This would provide precious ground information towards understanding the viral pathogenecity and protein level accumulation for early diagnosis of virus.

Molecular detection, identification, and sequence analysis of 'Candidatus Liberibacter asiaticus' associated with Huanglongbing disease of citrus in North India.

Huanglongbing (HLB, Citrus greening), caused by a phloem-limited fastidious gram-negative bacterium, "Candidatus Liberibacter spp.", is one of the devastating diseases of citrus worldwide. The pathogen belongs to the alpha-proteobacteria group and is classified on the basis of its geographical origin and 16S rRNA sequence diversity. Although the disease has been reported from all citrus growing states of India, the status and the molecular variability among the isolates from the Northern part of the country is unknown. A total of five different HLB isolates originating from Northern India showing variable symptoms were studied. The genomic regions of four different genes, i.e., 16S rRNA, intergenic 16S/23S rRNA spacer region, rplA-rplJ, and CLIBASIA_01645 were amplified by PCR, sequenced, and variations in these sequences were assessed. Analysis of 16S rRNA clearly indicated that all five isolates fit in to 'Candidatus Liberibacter asiaticus' (CLas) group. However, 16S/23S rRNA intergenic spacer region-based analysis failed to segregate these isolates beyond species level. Sequence analysis of rplA-rplJ gene and CLIBASIA_01645 loci also confirmed the existence of diversity among the 'CLas' in the surveyed areas. Further, 16S rRNA and rplA-rplJ-based SNP analysis revealed that some isolates segregated into three new lineages, two on the basis of 16Sr (16Sr-XV and 16Sr-XVI), and one based on ß-rp (rp-IV), respectively. A tandem repeat number (TRN) at CLIBASIA_01645 region were TRN = 5, 6 and 13; with TRN = 6 being common in three 'CLas' isolates. Overall, the study demonstrated that all examined five HLB isolates belonged to 'CLas' group. However, these isolates showed distinct sequence variability in three out of four genomic regions. The results provide a robust framework for understanding differences in pathogenicity among different HLB isolates as it is plausibly related to their genomic variation, and evolutionary history.

Characterization of the heavy metal binding properties of periplasmic metal uptake protein CLas-ZnuA2.

Candidatus Liberibacter asiaticus (CLas), a phloem-limited unculturable Gram-negative bacterium, causes citrus greening disease. The proteome analysis of CLas showed the presence of a heavy metal permease and Co/Zn/Cd cation exporter system. However, there is no designated metal uptake protein specific for the heavy metal permease in CLas. One of the metal uptake proteins, designated as CLas-ZnuA2, in our previous studies, showed a lower metal-binding affinity for Mn2+ and Zn2+ and was postulated to bind and transport metals rather non-specifically. The present study focused on the characterization of the heavy metal binding properties of CLas-ZnuA2 using SPR, CD, DSC and crystallographic studies. The crystal structure analysis of Cd2+ bound CLas-ZnuA2 showed octahedral geometry for Cd2+ binding as compared to a non-preferred square-pyramidal geometry for Mn2+ and Zn2+ binding in earlier reported crystal structures. In SPR analysis, the binding affinities of 4.7 × 10-6 M, 7.2 × 10-6 M, 5.3 × 10-5 M and 4.3 × 10-5 M for Hg2+, Cd2+, Ba2+ and Co2+ respectively were higher as compared to earlier reported values for Mn2+ and Zn2+. Likewise, CD and DSC analysis showed relatively higher thermal stability for CLas-ZnuA2 on heavy metal binding. Taken together with the expression of the permease and exporter system for heavy metals, our results indicate that CLas-ZnuA2 may be involved in sequestering and transport of various transition divalent metals in environmentally stressed conditions.

Mutation studies and structure-based identification of potential inhibitor molecules against periplasmic amino acid binding protein of Candidatus Liberibacter asiaticus (CLasTcyA).

Earlier reported crystal structure of CLasTcyA revealed unique features like relatively a larger substrate binding pocket, an extended C-terminal loop restricted by a disulfide bond and involvement of residues from hinge region in substrate binding. In present study, CLasTcyA mutants were created to evaluate the importance of these unique features through biophysical characterization. The Val58 in CLasTcyA was replaced by Trp, conserved in most cystine binding proteins, to reduce the size of the binding pocket. All other mutations were created in CLasTcyAV58W mutant as the presence of Trp could be used for intrinsic fluorescence studies. The CLasTcyAV58W showed a noticeable increase in binding affinity and thermal stability as compared to the native form. The mutation of two cysteines in triple mutant CLasTcyAV58W/C212S/C239S, removal of C-terminal extended loop in truncated CLasTcyAV58W/C212S and mutation of His95 from hinge region in the double mutant CLasTcyAV58W/H95A showed a marked decrease in stability-indicating the importance of the unique features in structure of CLasTcyA. The bioinformatics-based virtual screening was employed to screen the potential inhibitor molecules for detailed future studies. The results clearly establish the importance of unique features in structure-function relationship of CLasTcyA.

Rapid and Sensitive Detection of Citrus tristeza virus Using Reverse Transcription Loop-Mediated Isothermal Amplification (RT-LAMP) Assay.

Loop-mediated isothermal amplification (LAMP) is one recently developed gene amplification technique that emerges as a simple and quick diagnostic tool for early detection of nucleic acid targets. The LAMP technique works on the principle of strand displacement activity of Bst polymerase. It contains a set of four specially designed primers, which recognizes six different regions on the target nucleotide sequence. In the LAMP reaction, amplification is carried out in an isothermal conditions (60-65°C) using simple and inexpensive device like water bath or dry bath. Additional benefits of LAMP technique are that final results can be seen directly with naked eyes by adding intercalating dye SYBR Green I in the reaction tube. Reverse transcription loop-mediated isothermal amplification (RT-LAMP) is one of the novel techniques used for detection of RNA targets. The technology has been successfully applied for rapid and sensitive detection of Citrus tristeza virus (CTV) by using four oligo-primers, targeting a conserved coat protein gene (CPG) of an Indian CTV isolate. The result of assay is visible in naked eyes easily in the presence of SYBR Green I (100×) or on 1.5% agarose gel electrophoresis. CTV-RT-LAMP could be used away from plant pathology laboratories even in remote location.

Crystal structures of a putative periplasmic cystine-binding protein from Candidatus Liberibacter asiaticus: insights into an adapted mechanism of ligand binding.

The amino acid-binding receptors, a component of ABC transporters, have evolved to cater to different specificities and functions. Of particular interest are cystine-binding receptors, which have shown broad specificity. In the present study, a putative periplasmic cystine-binding protein from Candidatus Liberibacter asiaticus (CLasTcyA) was characterized. Analysis of the CLasTcyA sequence and crystal structures in the ligand-bound state revealed novel features of CLasTcyA in comparison to related proteins. One of the unique features found in CLasTcyA structure was the positioning of the C-terminal extended loop of one chain very close to the substrate-binding site of the adjacent monomer in the asymmetric unit. The presence of a disulphide bond, unique to Candidatus Liberibacter family, holds the C-terminal extended loop in position. Analysis of the substrate-binding pocket of CLasTcyA suggested a broad specificity and a completely different orientation of the bound substrates in comparison to related protein structures. The open conformation for one of the two chains of the asymmetric unit in the Arg-bound structure revealed a limited open state (18.4°) for CLasTcyA as compared to open state of other related proteins (~ 60°). The strong interaction between Asp126 on helix-α5 of small domain and Arg82 (bigger domain) restricts the degree of opening in ligand-free open state. The dissociation constant of 1.26 µm by SPR and 3.7 µm by MST exhibited low affinity for the cystine. This is the first structural characterization of an l-cystine ABC transporter from plant pathogen and our results suggest that CLasTcyA may have evolved to cater to its specific needs for its survival in the host.