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<p>A fluorescence imaging and real-time quantitative PCR (qPCR) approach was established to enable both qualitative and quantitative assessment of <i>Magnaporthe oryzae</i> colonization on rice leaf surfaces. Stable GFP-expressing transformants were generated using <i>Agrobacterium tumefaciens</i>-mediated transformation within 10–12&#xa0;days and confirmed by PCR with gene-specific primers. Expression of the gfp-gene was validated in planta using epifluorescence and confocal laser scanning microscopy. These transformants facilitated visualization of fungal colonization patterns on rice leaves over time. For quantitative estimation, a qPCR assay targeting the gfp-transgene was standardized, achieving a detection sensitivity of 0.3 femtograms of DNA. Absolute quantification showed an increase in pathogen biomass from 1.1 × 10<sup>2</sup> to 2.8 × 10<sup>4</sup> within 48&#xa0;h post-inoculation on both rice and wheat leaves. This integrated strategy, combining gfp-based imaging with qPCR, offers a reliable tool for tracking <i>Magnaporthe oryzae</i> colonization on the phylloplane and holds potential for application in host resistance screening and pathogenesis research.</p>

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Integrated fluorescence microscopy and qPCR analysis of pre-penetrative colonization of rice leaves by gfp-tagged Magnaporthe oryzae

  • 期刊论文

  • 发表日期：

    2025年5月9日

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  • Aundy Kumar,
  • Rohini Chawla,
  • Mushineni Ashajyothi,
  • Prabhakaran Narayanaswamy,
  • Vibhuti Munjal,
  • Neelam Sheoran

摘要

A fluorescence imaging and real-time quantitative PCR (qPCR) approach was established to enable both qualitative and quantitative assessment of Magnaporthe oryzae colonization on rice leaf surfaces. Stable GFP-expressing transformants were generated using Agrobacterium tumefaciens-mediated transformation within 10–12 days and confirmed by PCR with gene-specific primers. Expression of the gfp-gene was validated in planta using epifluorescence and confocal laser scanning microscopy. These transformants facilitated visualization of fungal colonization patterns on rice leaves over time. For quantitative estimation, a qPCR assay targeting the gfp-transgene was standardized, achieving a detection sensitivity of 0.3 femtograms of DNA. Absolute quantification showed an increase in pathogen biomass from 1.1 × 102 to 2.8 × 104 within 48 h post-inoculation on both rice and wheat leaves. This integrated strategy, combining gfp-based imaging with qPCR, offers a reliable tool for tracking Magnaporthe oryzae colonization on the phylloplane and holds potential for application in host resistance screening and pathogenesis research.