scholarly journals Editing of PCNA Gene by CRISPR/Cas9 Technology to Improve the Red Chili Resistance to Yellow Leaf Curl Disease

2020 ◽  
Vol 16 (2) ◽  
pp. 79
Author(s):  
Devi Ayu Kurniawati ◽  
NFN Suharsono ◽  
Tri Joko Santoso
Keyword(s):  
Germplasm Collection ◽  
Chili Pepper ◽  
Nuclear Antigen ◽  
Yellow Leaf ◽  
In Planta ◽  
Virus Inoculation ◽  
Gene Target ◽  
Transformed Plants ◽  
Leaf Curl Disease ◽  
Leaf Curl

<p>Yellow leaf curl disease caused by Pepper yellow leaf curl virus (PepYLCV), member of geminiviruses group, is responsible for heavy yield losses for chili pepper production. Resistant genes which can cause immunity to the disease have not been found in germplasm collection. The aim of the research was to edit proliferating cell nuclear antigen (PCNA) gene by using CRISPR/Cas9 technology for developing plant resistance against geminivirus in chili pepper. A CRISPR/Cas9 plasmid cassette construct harboring the guide RNA of PCNA gene was constructed by Golden Gate cloning strategy. The construct was then introduced into chili genome via in planta method using Agrobacterium tumefaciens EHA105. The transformed plants were bioassayed by virus inoculation and confirmed using PCR and DNA sequencing to identify a mutagenesis event in PCNA gene target. The results showed that CRISPR/Cas9 plasmid cassette harboring gRNA of PCNA gene was successfully constructed. In planta transformation using A. tumefaciens vector harboring CRISPR/Cas9-gRNA PCNA construct resulted in 307 and 193 transformed plants from chili var. Lingga and Ciko, respectively. Bioassay by using virus inoculation to the transformed plants obtained 6 and 14 lines of Lingga and Ciko, respectively, which were resistant to geminivirus (no symptom observed). The resistant lines of chili pepper var. Lingga and Ciko were mutated in PCNA gene with one base insertion or deletion mutation types. These results exhibit that the CRISPR/Cas9 genome editing can be used to induce mutant of PCNA gene in chili pepper. Further investigation is necessary to evaluate the selected chili lines resistant to PepYLCV infection.</p>

scholarly journals Resistance Analysis of CRISPR/Cas9 Genome-Edited Chili M2 Mutant Lines against Pepper Yellow Leaf Curl Viral Disease

2021 ◽  
Vol 17 (1) ◽  
pp. 1
Author(s):  
Wandy Murti Prasetya ◽  
Toto Hadiarto ◽  
Wening Enggarini ◽  
Aqwin Polosoro ◽  
Suharsono Suharsono
Keyword(s):  
Viral Disease ◽  
Chili Pepper ◽  
Nuclear Antigen ◽  
Pcr Analysis ◽  
Yellow Leaf ◽  
Resistant Lines ◽  
Mutant Lines ◽  
Leaf Curl Virus ◽  
Proliferating Cell ◽  
Leaf Curl

<p>Pepper yellow leaf curl virus (PepYLCV) infection transmitted by silverleaf whitefly (Bemisia tabaci [Gennadius]) can decrease chili pepper yield up to 100%. At this moment, there is no chili pepper variety resistant to PepYLCV available. Genome editing approach through CRISPR/Cas9 is an effort to develop variety resistance to the viral infection. The purpose of this study was to obtain M2 lines developed by CRISPR/Cas9 system on proliferating cell nuclear antigen (PCNA) gene for resistance to PepYLCV. A total of four M2 lines (C47-7, L84-2, L84-23, and L120-19) consisting of 60 chili plants were tested for their resistance to PepYLCV. PCR analysis was performed to detect the presence (infection) of the virus. The results showed that a total of 35 plants derived from the four lines were resistant to PepYLCV. They consisted of 7 plants from C47-7 line, 11 plants from L84-2 line, 9 plants from L84-23 line, and 8 plants from L120-19 line. PCR analysis confirmed that the resistant plants obtained from this study were negatively infected by the virus. Since not all tested plants were resistant to virus infection, the PCNA gene allele in these resistant lines were most likely heterozigotes. Sequencing of PCNA gene of the resistant lines is needed to confirm that the resistance phenotypes obtained was due to mutation of the gene. Therefore, further selection needs to be performed to obtain stable and PepYLCV-resistant lines.</p>

scholarly journals MOLECULAR IDENTIFICATION OF Pepper yellow leaf curl Indonesia virus ON CHILI PEPPER IN NUSA PENIDA ISLAND

2021 ◽  
Vol 21 (2) ◽  
pp. 97-102
Author(s):  
Dewa Gede Wiryangga Selangga ◽  
Listihani Listihani
Keyword(s):  
Sequence Analysis ◽  
Molecular Identification ◽  
Disease Incidence ◽  
Analysis Data ◽  
Chili Pepper ◽  
Yellow Leaf ◽  
Degenerate Primers ◽  
Disease Symptoms ◽  
Leaf Curl Disease ◽  
Leaf Curl

Molecular identification of Pepper yellow leaf curl Indonesia virus on chili pepper in Nusa Penida Island. Pepper yellow leaf curl Indonesia virus (PYLCV) has been reported as caused yellow leaf curl disease in Bali Island since early 2012. Dominant symptoms of PYLCV infection in chili pepper were yellowing, leaf curl, yellow mosaic, and mottle. Bemisia tabaci, has been known to vector on the case yellow leaf curl disease. Observations on the Nusa Penida Island in 2020 showed symptoms such as yellow leaf curl disease, however, identification of PYLCV in Nusa Penida Island has not been studied. Molecular identification was conducted using polymerase chain reaction and sequence analysis. Data collected in this study was disease symptoms and disease incidence. The results showed that dominant disease symptoms caused by virus from Nusa Penida were yellow mosaic, yellowing, and mottle. Universal DNA fragments of 912 bp were successfully amplified from 50 leaf samples using Begomovirus degenerate primers SPG 1 (5’-CCCCKGTGCGWRAATCCAT-3’) and SPG 2 (5’ATCCVAA YWTYCAGGGAGCT-3’). Sequence analysis showed that the isolate from Nusa Penida was a Pepper yellow leaf curl Indonesia virus with a 98–100% homology with several reference isolates.

scholarly journals Potency of Yeasts to Promote Induced Resistance on Chili Plant (Capsicum annuum L.) against Yellow Leaf Curl Disease

2020 ◽  
Vol 24 (1) ◽  
pp. 34
Author(s):  
Sri Hartati ◽  
Rika Meliansyah ◽  
Luciana Djaya ◽  
Ceppy Nasahi ◽  
Ramadhan Kamal Putra
Keyword(s):  
Salicylic Acid ◽  
Incubation Period ◽  
Induced Resistance ◽  
Peroxidase Activity ◽  
Disease Incidence ◽  
Yellow Leaf ◽  
Virus Inoculation ◽  
Control Virus ◽  
Leaf Curl Disease ◽  
Leaf Curl

Yellow leaf curl is a major disease on chili plants. The use of antagonistic yeasts as the control agents is a good alternative in  an environmentally friendly control method. This study was objected to evaluate the potencies of Rhodotorula minuta and Candida tropicalis to promote induced resistance on chili plants against yellow leaf curl disease. The experiment was arranged in the randomized complete block design with 9 treatments and 3 replications. The treatments were the application of the yeasts at three different times of virus inoculation, as follows: A. R. minuta, virus inoculation at 3 days after transplanting (dat), B. R. minuta, virus inoculation at 7 dat, C. R. minuta, virus inoculation at 10 dat, D. C. tropicalis, virus inoculation at 3 dat, E. C. tropicalis, virus inoculation at 7 dat, F. C. tropicalis, virus inoculation at 10 dat, G. control, virus inoculation at 3 dat, H. control, virus inoculation at 7 dat, and I. control, virus inoculation at 10 dat. No treatment was applied to the control.  The yeasts were applied by soaking the chili seeds, and pouring the suspension into the growth media at transplanting. The variables observed were incubation period, disease severity, and disease incidence. Peroxidase activity, phenylalanine ammonia lyase activity, and salicylic acid accumulation were also analyzed. The results showed that the application of R. minuta or C. tropicalis was able to extend the disease incubation period, but did not reduce the yellow leaf curl disease incidence and severity. The treatment of R. minuta, virus inoculation at 7 dat, increased the peroxidase activity from 2590.80 units to 6870.93 units (0.5 minute) and from 577.367 units to 1131.300 units (2.5 minutes), PAL activity from 16.059 to 17.911 A290/mg, and accumulation of salicylic acid from 2.785 to 6.263 ppm. Application of C. tropicalis, virus inoculation at 7 dat, increased the peroxidase activity from 2590.80 units to 6033.067 units (0.5 minute) and from 577.367 units to 950.967 units (2.5 minutes), and accumulation of salicylic acid from 2.785 to 6.982 ppm.

scholarly journals The Effect of Silica (SiO2) to the Severity of Yellow Leaf Curl Disease on Chili Pepper

2019 ◽  
Vol 23 (1) ◽  
pp. 54
Author(s):  
Dewa Gede Wiryangga Selangga ◽  
Sri Hendrastuti Hidayat ◽  
Anas Dinurrohman Susila ◽  
Suryo Wiyono
Keyword(s):  
Disease Severity ◽  
Negative Impact ◽  
Disease Incidence ◽  
Block Design ◽  
Total Content ◽  
Chili Pepper ◽  
Yellow Leaf ◽  
Severity Of The Disease ◽  
Leaf Curl Disease ◽  
Leaf Curl

Yellow leaf curl disease of chili pepper caused by Pepper yellow leaf curl virus (PYLCV) has been reported as an important disease in Java and Bali. Disease severity reached 80−100% and it may cause significant yield losses. In order to reduce the negative impact of synthetic insecticides, silica application was evaluated for its potency to suppress the disease. A greenhouse experiment was conducted using randomly block design with 2 factors: PYLCV isolate (Java and Bali) and silica (SiO2) treatment (with and without). Parameters observed were disease symptoms, incubation period, disease incidence and severity, and total silica level. The symptoms of virus infection in Pelita 8 and Seret cultivars were yellow mosaic, leaf curl, green mosaic, dwarf, and cupping upward or downward. A Specific DNA fragment of 912 bp was successfully amplified from 4 samples. Four sequences were obtained and further analysis showed their highest homology, i.e. 96% and 97% with Pepper yellow leaf curl Indonesia virus-Java (PYLCIV-Java) (JX416180) and PYLCIV-KrthAl (LC381274), respectively. Infection by different virus isolates did not affect disease severity significantly. The application of silica was able to delay symptom development and to suppress the severity of the disease in the range of 16.67−30.33%. Silica application on the soil increased the total content of silica in the plants. However, a further experiment is required to understand the mode of action of silica in inducing plant resistance to the pathogen.

Mapping of QTLs in tomato line FLA456 associated with resistance to a virus causing tomato yellow leaf curl disease

Euphytica ◽  
2012 ◽  
Vol 190 (2) ◽  
pp. 297-308 ◽  
Author(s):  
P. Kadirvel ◽  
R. de la Peña ◽  
R. Schafleitner ◽  
S. Huang ◽  
S. Geethanjali ◽  
...  
Keyword(s):  
Tomato Yellow Leaf ◽  
Yellow Leaf ◽  
Tomato Line ◽  
Leaf Curl Disease ◽  
Leaf Curl

scholarly journals Engineering Plant Resistance to Tomato Yellow Leaf Curl Thailand Virus Using a Phloem-Specific Promoter Expressing Hairpin RNA

2020 ◽  
Vol 33 (1) ◽  
pp. 87-97
Author(s):  
Yuh Tzean ◽  
Ho-Hsiung Chang ◽  
Tsui-Chin Tu ◽  
Bo-Han Hou ◽  
Ho-Ming Chen ◽  
...  
Keyword(s):  
Chlorophyll Synthesis ◽  
Tomato Yellow Leaf ◽  
Cauliflower Mosaic Virus ◽  
Rice Tungro Bacilliform Virus ◽  
35S Promoter ◽  
Yellow Leaf ◽  
Hairpin Rna ◽  
Leaf Curl Disease ◽  
Inoculation Assay ◽  
Leaf Curl

Transgenic approaches employing RNA interference (RNAi) strategies have been successfully applied to generate desired traits in plants; however, variations between RNAi transgenic siblings and the ability to quickly apply RNAi resistance to diverse cultivars remain challenging. In this study, we assessed the promoter activity of a cauliflower mosaic virus 35S promoter (35S) and a phloem-specific promoter derived from rice tungro bacilliform virus (RTBV) and their efficacy to drive RNAi against the endogenous glutamate-1-semialdehyde aminotransferase gene (GSA) that acts as a RNAi marker, through chlorophyll synthesis inhibition, and against tomato yellow leaf curl Thailand virus (TYLCTHV), a begomovirus (family Geminiviridae) reported to be the prevalent cause of tomato yellow leaf curl disease (TYLCD) in Taiwan. Transgenic Nicotiana benthamiana expressing hairpin RNA of GSA driven by either the 35S or RTBV promoter revealed that RTBV::hpGSA induced stronger silencing along the vein and more uniformed silencing phenotype among its siblings than 35S::hpGSA. Analysis of transgenic N. benthamiana, 35S::hpTYLCTHV, and RTBV::hpTYLCTHV revealed that, although 35S::hpTYLCTHV generated a higher abundance of small RNA than RTBV::hpTYLCTHV, RTBV::hpTYLCTHV transgenic plants conferred better TYLCTHV resistance than 35S::hpTYLCTHV. Grafting of wild-type (WT) scions to TYLCTHV RNAi rootstocks allowed transferable TYLCTHV resistance to the scion. A TYLCTHV-inoculation assay showed that noninfected WT scions were only observed when grafted to RTBV::hpTYLCTHV rootstocks but not 35S::hpTYLCTHV nor WT rootstocks. Together, our findings demonstrate an approach that may be widely applied to efficiently confer TYLCD resistance.

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