Utility of a tissue culture-independent Agrobacterium-mediated in planta transformation strategy in bell pepper to develop fungal disease resistant plants

Arthikala, Manoj‐Kumar; Nanjareddy, Kalpana; Lara, Miguel; Sreevathsa, Rohini

doi:10.1016/j.scienta.2014.02.034

Cited by 11 publications

(9 citation statements)

References 38 publications

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“…4c ). We also examined changes in the expression of defense-related genes including SA-responsive CaPR1 and CaNPR1 43 , JA-responsive CaDEF1 , and CaWRKY40 in the transient expression system. The results showed that the relative transcript levels of CaPR1 , CaNPR1 , CaDEF1 , and CaWRKY40 increased continuously during transient expression of CaCDPK15 .…”

Section: Resultsmentioning

confidence: 99%

CaCDPK15 positively regulates pepper responses to Ralstonia solanacearum inoculation and forms a positive-feedback loop with CaWRKY40 to amplify defense signaling

Shen

Yang

et al. 2016

Sci Rep

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CaWRKY40 is a positive regulator of pepper (Capsicum annum) response to Ralstonia solanacearum inoculation (RSI), but the underlying mechanism remains largely unknown. Here, we functionally characterize CaCDPK15 in the defense signaling mediated by CaWRKY40. Pathogen-responsive TGA, W, and ERE boxes were identified in the CaCDPK15 promoter (pCaCDPK15), and pCaCDPK15-driven GUS expression was significantly enhanced in response to RSI and exogenously applied salicylic acid, methyl jasmonate, abscisic acid, and ethephon. Virus-induced gene silencing (VIGS) of CaCDPK15 significantly increased the susceptibility of pepper to RSI and downregulated the immunity-associated markers CaNPR1, CaPR1, and CaDEF1. By contrast, transient CaCDPK15 overexpression significantly activated hypersensitive response associated cell death, upregulated the immunity-associated marker genes, upregulated CaWRKY40 expression, and enriched CaWRKY40 at the promoters of its targets genes. Although CaCDPK15 failed to interact with CaWRKY40, the direct binding of CaWRKY40 to pCaCDPK15 was detected by chromatin immunoprecipitation, which was significantly potentiated by RSI in pepper plants. These combined results suggest that RSI in pepper induces CaCDPK15 and indirectly activates downstream CaWRKY40, which in turn potentiates CaCDPK15 expression. This positive-feedback loop would amplify defense signaling against RSI and efficiently activate strong plant immunity.

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Section: Resultsmentioning

confidence: 99%

CaCDPK15 positively regulates pepper responses to Ralstonia solanacearum inoculation and forms a positive-feedback loop with CaWRKY40 to amplify defense signaling

Shen

Yang

et al. 2016

Sci Rep

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“…The apical meristem-targeted in planta transformation coupled with stringent kanamycin selection was used to develop chili transformants 35 . Thorough evaluation in successive generations identified five homozygous T 3 plants (16-1, 58-11, 41-5, 87-3 and 104-6) from 5 independent T 1 events as promising.…”

Section: Discussionmentioning

confidence: 99%

“…Five day-old germinated seedlings of chili (cultivar fof-2 ) were used as explants for in planta transformation 35 , 44 using Agrobacterium tumefaciens containing PDH45 . T 1 seeds from primary transformants were harvested and screened for the selection of putative transformants.…”

Section: Methodsmentioning

confidence: 99%

Overexpression of Pea DNA Helicase 45 (PDH45) imparts tolerance to multiple abiotic stresses in chili (Capsicum annuum L.)

Shivakumara

Sreevathsa

Dash

et al. 2017

Sci Rep

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Imparting tolerance to abiotic stresses is of global importance as they inflict significant yield losses in field as well as in vegetable crops. Transcriptional activators, including helicases are identified to play a pivotal role in stress mitigation. Helicases, also known as molecular motors, are involved in myriad cellular processes that impart intrinsic tolerance to abiotic stresses in plants. Our study demonstrates the potential of a Pea DNA Helicase 45 (PDH45), in combating multiple abiotic stresses in chili. We harnessed Agrobacterium-mediated in planta transformation strategy for the generation of stable, single copy transgenic events. Precise molecular detection of the transgenes by sqRT-PCR coupled with genomic Southern analysis revealed variation in the integration of PDH45 at distinct loci in independent transgenic events. Characterization of five promising transgenic events showed both improved response to an array of simulated abiotic stresses and enhanced expression of several stress-responsive genes. While survival and recovery of transgenic events were significantly higher under gradual moisture stress conditions, under imposition of moderate stress, the transgenic events exhibited invigorated growth and productivity with concomitant improvement in water use efficiency (WUE). Thus, our study, unequivocally demonstrated the cardinal role of PDH45 in alleviating multiple abiotic stresses in chili.

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“…Regarding the high impact of the Agrobacterium infected tissue type on the success of this technique, we used two different tissues, the shoot apical meristem and ovary. Various plant tissues and organs such as microspores, seeds, internodes, and shoot apical meristems have been utilised by researchers for the in planta transformation method (Arthikala et al 2014). However, most researchers used SAM for the generation of stable transgenic plants in this system regarding the fact that undifferentiated cells can normally change to differentiated cells and regenerate into plants.…”

Section: Discussionmentioning

confidence: 99%

The application of an Agrobacterium-mediated in planta transformation system in a Catharanthus roseus medicinal plant

Bahari¹,

Sazegari²,

Niazi‎³

et al. 2020

CZECH J GENET PLANT

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The lack of an efficient protocol for the transformation of Catharanthus roseus, as an important medicinal plant, causes obstacles in the genetic engineering programmes of this plant. Therefore, the present study was conducted to examine a convenient and reliable system for generating stable transgenic lines based on the in planta method. To do so, two different in planta Agrobacterium infection systems were investigated. The transgenic lines were produced through ovary injection and shoot apical meristem (SAM) immersing methods. The presence and expression of the GFP transgene in the T0 and T1 lines were confirmed by PCR and RT-PCR. Moreover, the ELISA test confirmed the GFP expression in the transgenic lines. Apart from the superiority of the ovary injection over the SAM inoculation due to the normal growth and healthy appearance, it also showed a significant difference from the point of the confirmed transgenic plant numbers. GFP integration was confirmed in 6 out of 50 T1 plants from the ovary injection and only 3 out of 150 plants generated from the SAM inoculation. In addition to the comparison of these two separate methods, we reached a new convenient in planta system by a 12% transformation rate for generating the C. roseus transgenic lines. According to the result of this study, ovary injection can be introduced as a novel, facile, and stable method for the Catharanthus transformation programme.

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Utility of a tissue culture-independent Agrobacterium-mediated in planta transformation strategy in bell pepper to develop fungal disease resistant plants

Cited by 11 publications

References 38 publications

CaCDPK15 positively regulates pepper responses to Ralstonia solanacearum inoculation and forms a positive-feedback loop with CaWRKY40 to amplify defense signaling

CaCDPK15 positively regulates pepper responses to Ralstonia solanacearum inoculation and forms a positive-feedback loop with CaWRKY40 to amplify defense signaling

Overexpression of Pea DNA Helicase 45 (PDH45) imparts tolerance to multiple abiotic stresses in chili (Capsicum annuum L.)

The application of an Agrobacterium-mediated in planta transformation system in a Catharanthus roseus medicinal plant

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