Rapid Detection and Quantification of Plant Innate Immunity Response Using Raman Spectroscopy.

Chung-Hao Huang,Rajani Sarojam,Jun Sung Seo,Rajeev J Ram,Gajendra P Singh,Nam-Hai Chua,Hui-Zhu Mao,Pil Joong Chung,Piyarut Diloknawarit,Sayuj Koyyappurath

doi:10.3389/fpls.2021.746586

Abstract

We have developed a rapid Raman spectroscopy-based method for the detection and quantification of early innate immunity responses in Arabidopsis and Choy Sum plants. Arabidopsis plants challenged with flg22 and elf18 elicitors could be differentiated from mock-treated plants by their Raman spectral fingerprints. From the difference Raman spectrum and the value of p at each Raman shift, we derived the Elicitor Response Index (ERI) as a quantitative measure of the response whereby a higher ERI value indicates a more significant elicitor-induced immune response. Among various Raman spectral bands contributing toward the ERI value, the most significant changes were observed in those associated with carotenoids and proteins. To validate these results, we investigated several characterized Arabidopsis pattern-triggered immunity (PTI) mutants. Compared to wild type (WT), positive regulatory mutants had ERI values close to zero, whereas negative regulatory mutants at early time points had higher ERI values. Similar to elicitor treatments, we derived an analogous Infection Response Index (IRI) as a quantitative measure to detect the early PTI response in Arabidopsis and Choy Sum plants infected with bacterial pathogens. The Raman spectral bands contributing toward a high IRI value were largely identical to the ERI Raman spectral bands. Raman spectroscopy is a convenient tool for rapid screening for Arabidopsis PTI mutants and may be suitable for the noninvasive and early diagnosis of pathogen-infected crop plants.

Highlights

Global food supply and security have been challenged by water scarcity and climate change, and the situation is further exacerbated by the projected increase in the world’s population to about 10 billion by 2050 (Misra, 2014)
To reduce variability between biological samples, Arabidopsis wild type (WT) (Col-0) and mutant plants were grown under short-day conditions for about 5–6 weeks with about 17–18 leaves (Farmer et al, 2013; Kurenda et al, 2019) (Figure 1a)
The abaxial side of leaf seventh or eighth was infiltrated with a needleless syringe containing (50 μl) water, 1.0 μM flg22, or 1.0 μM elf18 (Figure 1b)

Summary

Introduction

Global food supply and security have been challenged by water scarcity and climate change, and the situation is further exacerbated by the projected increase in the world’s population to about 10 billion by 2050 (Misra, 2014). Over the last two decades, plant disease diagnosis has shifted from a visual inspection of symptom-based disease phenotype to molecular-based diagnostic procedures (Fang and Ramasamy, 2015; Martinelli et al, 2015). These molecular methods suffer from several drawbacks such as a dependency on the availability of pathogen-specific gene sequences or antibodies. Their implementation requires time and is not amenable to on-site field application

Methods

Results

Conclusion