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ThesisItem Open Access Development of recombinant coat protein for immunodiagnosis of banana bunchy top and bract mosaic diseases(Department of Plant Pathology, College of Agriculture, Vellanikkara, 2021) Darsana Dilip, K C; KAU; Vimi, LouisThe present investigation was undertaken to develop recombinant coat protein (rCP) of Banana bunchy top virus (BBTV) and Banana bract mosaic virus (BBrMV) for immunodetection of the viruses. The experiments were conducted at the Virology Lab, Banana Research Station, Kannara; Department of Plant Pathology, College of Agriculture, Vellanikkara, Kerala Agricultural University and Indian Institute of Science, Bengaluru during the period of 2016-2020. A roving survey in 10 districts of Kerala, divided into population subsets viz., North, Central and Southern zones were conducted for sample collection. After a preliminary DAC-ELISA, 17 and 12 representative samples respectively were selected and carried forward for further evaluations. The CP gene of BBTV was amplified from the total DNA isolated using reported primers by Polymerase Chain Reaction (PCR) and that of BBrMV by Reverse Transcriptase-PCR (RT-PCR). The CP gene sequences of these isolates were determined and submitted in the NCBI-GenBank Database. The 17 BBTV isolates were designated as MT174314-MT174330 and the 12 BBrMV isolates as MT818176- MT818187. It was inevitable to evaluate the molecular diversity of the viruses prior to devising nucleic- acid based and serological detection methods. The phylogeographic analysis depicted a clear demarcation of BBTV Kerala isolates based on geography whereas no such clustering was observed in the case of BBrMV isolates. Being an RNA virus, the molecular diversity of BBrMV (ranging between 1-12 %) was higher than BBTV. However, the 5’ and 3’ terminal of BBrMV CP gene was hypervariable and found unsuitable to be targeted for nucleic-acid based detection. Hence, forward primer was designed from the NIb region of ssRNA genome of BBrMV and reverse primer from 3’ UTR region upstream and downstream to the CP gene respectively. For nucleic-acid based detection of BBTV, highly conserved non-coding region of DNA-S upstream and downstream to the CP ORF was targeted. The primers were validated by detecting virus from the field samples collected from various parts of the state. The rCPs were chosen as a potential antigen for raising antibodies in order to develop serodiagnostic assays for the early detection of the viruses. The BBTV CP gene was clonedin to three expression vectors viz., pRSET-C, pGEX-4T-2 and pET32a(+) and transformed to expression hosts like BL21 (DE3) pLysS, Rosetta (DE3) pLysS and C41 strains of E. coli after amplification in DH5α. The 20 kDa recombinant BBTV CP (rBBTV CP) cloned in to pRSET-C, and overexpressed in various E. coli hosts had a hexa histidine (6X His) tag at the N terminal. Similarly, a 37 kDa fusion protein (pET/rBBTV CP) was overexpressed from pET/BBTVCP clone had a thioredoxin (Trx) tag (17 kDa) along with the 6X His tag. Whereas, a 45 kDa fusion protein (pGEX/rBBTV CP) with GST tag was overexpressed from pGEX/BBTVCP clone. These affinity tags in the fusion rCP enabled purification from other E. coli proteins. Although pRSET/rBBTV CP was soluble, the 20 kDa protein was highly unstable and partially degraded during purification at 4 °C. Curiously, pGEX/rBBTV CP dissociated from its GST affinity tag and the rCP without the tag degraded. On evaluating the protease cleavage sites in the fusion protein, trypsin cleavage sites were present between the C terminal of GST and N terminal of BBTV CP which might be the reason for cleavage of the ~20 kDa protein from its affinity tag. Thus, it was impossible to purify the protein from the pool of E. coli proteins. Restriction free (RF) cloning of BBTV CP to pGEX-4T-2 was attempted not only to replace these trypsin cleavage sites but also the thrombin cleavage site present in the vector with Tobacco etch virus (TEV) NIa protease site. Thrombin is a specific enzyme used to cleave off the tag from the fusion protein after purification. However, its specificity is not universal. Furthermore, the commercially available enzyme is costly. TEV protease on other hand was produced in the laboratory and was highly specific. However, the cleavage using TEV protease was unsuccessful apparently because of a steric hindrance contributed by the two extremely ordered regions flanking the TEV cleavage site present in the disordered region of the fusion protein. pET/rBBTV CP was highly soluble like ΔpGEX/rBBTVCP. Likewise, BBrMV CP gene was cloned into pRSET-C and pGEX-4T-2 to obtain pRSET/rBBrMV CP and pGEX/rBBrMV CP of size 34 kDa and 60 kDa respectively. The 34 kDa pRSET/rBBrMV CP was insoluble. Overexpression and purification of the protein was standardized in various conditions to increase solubility. On the contrary, pGEX/rBBrMV CP was highly soluble and was purified by GSH Sepharose affinity column chromatography. 360 μg/ml of untagged protein was obtained from 1 l culture. However, like any other potyviral CP, the exposed N and C terminal of BBrMV CP was also prone to proteolytic cleavage. It partially degraded when incubated with thrombin atroom temperature for GST tag cleavage. All these bands were detected by potyviral CP specific antibody in Western blot. Further on storage complete degradation of the protein was observed. Further standardisation of the protocol is necessary to either stabilise monomeric CP or develop BBrMV VLPs in vitro for immunising animal in order to raise the antiserum. The immunogenicity of the antigens (rBBTV CP and rBBrMV CP) was confirmed by Western blot using BBTV CP specific and potyvirus CP specific antibody procured from NRC, Banana and IISc, Bangalore respectively. The rCPs were also characterized by fluorescence spectroscopy, sucrose gradient ultra centrifugation and electron microscopy. The fluorescent spectra of tagged and tag less rBBrMV CP deviated from 330 nm which is typical for a partially disordered protein. However, the spectra of pET/rBBTV CP and ΔpGEX/rBBTV CP were different. The former depicted the spectra of a mostly globular protein. There were two λmax for the fluorescence spectra of ΔpGEX/rBBTV CP. The epitope prediction of BBTV CP with Trx tag gave interesting insights. A single linear epitope of 80 residues were detected in pET/rBBTV CP comprising of C terminal of the affinity tag and the N terminal of BBTV CP. This was expected to increase the immunogenicity of the antigen and administered for production of antiserum. The titre value of polyclonal antiserum produced against the 37 kDa pET/rBBTV CP was evaluated by DAC-ELISA and was found to be 1:128000. Titre value for serological assays of field samples was standardized as 1:10000 to be more inclusive for detecting virus even at early stages of infection. A total of 247 tissue culture samples and 10 field samples were screened for the presence of the virus using the antiserum and was compared with the procured antiserum. Seemingly, the latter non-specifically reacted with plant proteins which gave a higher absorbance value in negative control and correspondingly high absorbance in the infected samples. The polyclonal antiserum raised against rBBTV CP was used to standardize serological detection assays like IC-PCR, DIBA and TAS-ELISA apart from DAC-ELISA. DIBA and TAS-ELISA were the most sensitive assays which could detect up to 1:80 dilution of the antigen. In conclusion, due to the higher nucleotide variability of the CP gene, serological detection is preferred over nucleic acid based assays. However, the quality of antigen used for raising the antibody plays a major role in serodiagnostics. Hence, high quality rCPs of both BBTV and BBrMV were developed in the laboratory in various vector/host systems. ThepET/rBBTV CP overexpressed in C41 strain of E.coli (1.1 mg/ ml obtained from 1 L culture) was used for immunisation of the animal. A highly sensitive antiserum specific to BBTV with a titre ten fold higher than that of the commercially available antiserum was obtained. Using this antiserum raised against rBBTV CP, various serodiagnostic assays were standardised in the laboratory. Among these, TAS-ELISA was the most sensitive, detecting antigen even at higher dilution.ThesisItem Open Access Characterisation and management of sugarcane bacilliform virus (SCBV) causing leaf fleck disease in sugarcane(Department of Plant Pathology, College of Agriculture, Vellanikkara, 2021) Sanju Balan; KAU; Anita CherianSugarcane (Saccharum officinarum) is a monocotyledonous perennial cash crop cultivated worldwide both under tropical and sub tropical conditions. It is being cultivated in more than 120 countries in the world. Like any other crops, it is also susceptible to biotic stress. Of which, diseases caused by viruses not only pose serious threat to sugarcane cultivation but also result in deterioration and exclusion of elite varieties of the germplasm. One of the major viral disease which affects global exchange of sugarcane germplasm is leaf fleck disease caused by Sugarcane bacilliform virus (SCBV). The research project entitled ‘Characterization and management of Sugarcane bacilliform virus causing leaf fleck in sugarcane’ was initiated with purposive sampling surveys in selected sugarcane fields in districts of Kerala and Tamil Nadu in order to document the symptoms under natural conditions, to assess the disease incidence, severity and to collect infected samples for further studies. The per cent disease incidence of the leaf fleck disease in Kerala ranged from 12 to 51 per cent whereas severity ranged from 10 to 36.5%. In Tamil Nadu the per cent disease incidence ranged from 28 to 56 per cent while severity ranged from 28 to 50.41%. Major symptoms observed on leaves were mottling, chlorotic flecks, chlorotic patches streaks and stripes with general yellowing of the canopy. In the case of severely affected clones, there was reduction in tillering, internodal length, number of internodes and appearance of deep longitudinal cracks. In highly susceptible clones, stunted growth with bunchy top appearance was noticed. On the basis of phenotypic variability of symptom expression, genotypes were classified into five groups. The development of the symptoms was also studied under artificial condition through insect transmission of the virus using pink mealy bug, Saccharicoccussacchari. Morphological characterisation of the virus done using electron microscopy revealed the presence of bacilliform virus particles of size 30 X 130–150 nm which indicated that the virus belongs to genus BADNA and family Caulimoviridae and the etiology of the disease was confirmed as Sugarcane bacilliform virus. The molecular detection of SCBV was also standardized through polymerase chain reaction (PCR). PCR amplification of RNaseH/RT gene was done using BADNA specific and SCBV129 specific primers. The amplicons were sequenced and in silco analysis of sequences showed sequence homology of 99 to 100 percent identity to SCBV. Widespread occurrence of the disease was observed even in the early generation of varietal development and in newly developed varieties. The transmission of the virus was suspected through true seed (fluff) developed by biparental crossing during sugarcane varietal development programme. Hence, the study was conducted to establish possible transmission of the virus from sugarcane parents to their progenies and the role of maternal and paternal parents in disease transmission through true seeds to the progenies. Samples from eight months old seedlings, three months old seedlings and parental clones were tested positive to the virus in PCR assays. Real time PCR was also standardized to assay these clones. Immunodiagnostic technique was validated using DAC ELISA. The technique of immunocapture PCR was also standardized. Minimal dilution of antisera with which SCBV could be detected was 2:1000 (V/V). Plant extract (antigen) at a dilution of 1:5 was found to be optimal for the detection of SCBV. Molecular detection of SCBV from mealy bug vector was also standardized. Both phenotypic and molecular methods were utilized to identify potential sources of natural resistance against SCBV. Based on the severity of symptom expression and PCR assays these were further classified as highly susceptible (HS), moderately susceptible (MS) moderately resistant (MR) and resistant (R). For generation of RNAi hair pin construct, initially forward (SF) and reverse primer (SR) were used to amplify 700 bp fragment of RT/RNase H gene to be cloned in sense orientation of the vector, pHANNIBAL. The linearized vector and the insert were ligated, and the ligation mixture was used to transform competent cells of Escherichia coli and the transformants were selected. Later antisense forward (AF) and reverse (AR) primer pairswere used to amplify 700 bp fragment of RT/RNase H gene to be cloned in antisense orientation. PCR product ligated into antisense direction of the vector and transformed into competent cells of E. coli. The recombinant pHANNIBAL vector was digested with restriction enzymes. The recombinant pHANNIBAL vector harbouringRNase H /RT gene was released from the vector through Not I site and subcloned into plant expression binary vector. Thus, cassette for RNA silencing was prepared.130 Meristem tip culture was also standardized with antiviral chemical tenofovir. Recovery percentage of meristem varied from 70 to 75 per cent and the viral load was quantified using real time PCR. The outcome of the study would facilitate early detection and elimination of the source of infection and prevent the spread of the disease in the field. Information generated in the study could be utilized while planning biparental crossing and reduce the spread of the virus in varietal development programmes. The hair pin construct developed in this study could be further utilized to generate transgenic disease resistant plants.
