AUTHOR=Agati Giovanni , Brunetti Cecilia , Tuccio Lorenza , Degano Ilaria , Tegli Stefania 

TITLE=Retrieving the in vivo Scopoletin Fluorescence Excitation Band Allows the Non-invasive Investigation of the Plant–Pathogen Early Events in Tobacco Leaves

JOURNAL=Frontiers in Microbiology

VOLUME=13

YEAR=2022

URL=https://www.frontiersin.org/journals/microbiology/articles/10.3389/fmicb.2022.889878

DOI=10.3389/fmicb.2022.889878

ISSN=1664-302X

ABSTRACT=<p>In this study, we developed and applied a new spectroscopic fluorescence method for the <italic>in vivo</italic> detection of the early events in the interaction between tobacco (<italic>Nicotiana tabacum</italic> L.) plants and pathogenic bacteria. The leaf disks were infiltrated with a bacterial suspension in sterile physiological solution (SPS), or with SPS alone as control. The virulent <italic>Pseudomonas syringae</italic> pv. <italic>tabaci</italic> strain ATCC 11528, its non-pathogenic <italic>ΔhrpA</italic> mutant, and the avirulent <italic>P. syringae</italic> pv. <italic>tomato</italic> strain DC3000 were used. At different post-infiltration time–points, the <italic>in vivo</italic> fluorescence spectra on leaf disks were acquired by a fiber bundle-spectrofluorimeter. The excitation spectra of the leaf blue emission at 460 nm, which is mainly due to the accumulation of coumarins following a bacterial infiltration, were processed by using a two-bands Gaussian fitting that enabled us to isolate the scopoletin (SCT) contribution. The pH-dependent fluorescence of SCT and scopolin (SCL), as determined by <italic>in vitro</italic> data and their intracellular localization, as determined by confocal microscopy, suggested the use of the longer wavelength excitation band at 385 nm of 460 nm emission (F<sub>385_460</sub>) to follow the metabolic evolution of SCT during the plant–bacteria interaction. It was found to be directly correlated (<italic>R</italic><sup>2</sup> = 0.84) to the leaf SCT content, but not to that of SCL, determined by HPLC analysis. The technique applied to the time-course monitoring of the bacteria–plant interaction clearly showed that the amount and the timing of SCT accumulation, estimated by F<sub>385_460</sub>, was correlated with the resistance to the pathogen. As expected, this host defense response was delayed after <italic>P. syringae</italic> pv. <italic>tabaci</italic> ATCC 11528 infiltration, in comparison to <italic>P. syringae</italic> pv. <italic>tomato</italic> DC3000. Furthermore, no significant increase of F<sub>385_460</sub> (SCT) was observed when using the non-pathogenic <italic>ΔhrpA</italic> mutant of <italic>P. syringae</italic> pv. <italic>tabaci</italic> ATCC 11528, which lacks a functional Type Three Secretion System (TTSS). Our study showed the reliability of the developed fluorimetric method for a rapid and non-invasive monitoring of bacteria-induced first events related to the metabolite-based defense response in tobacco leaves. This technique could allow a fast selection of pathogen-resistant cultivars, as well as the on-site early diagnosis of tobacco plant diseases by using suitable fluorescence sensors.</p>