CaCML13 Acts Positively in Pepper Immunity Against Ralstonia solanacearum Infection Forming Feedback Loop with CabZIP63

Lei Shen,Sheng Yang,Deyi Guan,Shuilin He

doi:10.3390/ijms21114186

Abstract

Ca2+-signaling—which requires the presence of calcium sensors such as calmodulin (CaM) and calmodulin-like (CML) proteins—is crucial for the regulation of plant immunity against pathogen attack. However, the underlying mechanisms remain elusive, especially the roles of CMLs involved in plant immunity remains largely uninvestigated. In the present study, CaCML13, a calmodulin-like protein of pepper that was originally found to be upregulated by Ralstonia solanacearum inoculation (RSI) in RNA-seq, was functionally characterized in immunity against RSI. CaCML13 was found to target the whole epidermal cell including plasma membrane, cytoplasm and nucleus. We also confirmed that CaCML13 was upregulated by RSI in pepper roots by quantitative real-time PCR (qRT-PCR). The silencing of CaCML13 significantly enhanced pepper plants’ susceptibility to RSI accompanied with downregulation of immunity-related CaPR1, CaNPR1, CaDEF1 and CabZIP63. In contrast, CaCML13 transient overexpression induced clear hypersensitivity-reaction (HR)-mimicked cell death and upregulation of the tested immunity-related genes. In addition, we also revealed that the G-box-containing CaCML13 promoter was bound by CabZIP63 and CaCML13 was positively regulated by CabZIP63 at transcriptional level. Our data collectively indicate that CaCML13 act as a positive regulator in pepper immunity against RSI forming a positive feedback loop with CabZIP63.

Highlights

Plants are frequently exposed to potentially pathogenic microbes and have evolved a sophisticated defense system initiate defense upon pathogen attack
We performed another experiment by quantitative real-time PCR to assay the transcript levels of this genes upon challenge of R. solanacearum inoculation (RSI) in the roots of pepper plants, the result showed that the transcript expression levels of this calmodulin like (CML) gene were enhanced from 3 h post-inoculation by RSI until 48 h post-inoculation with the peak observed at 12 h post-inoculation (Figure 1b), indicating that this CML was upregulated by RSI
This results, we performed another experiment by quantitative real-time PCR to assay the transcript levels of this genes upon challenge of R. solanacearum inoculation (RSI) in the roots of pepper plants, the result showed that the transcript expression levels of this CML gene were enhanced from 3 hInpt. oJ. sMto-li.nScoi.c2u02l0a, t2i1o, 4n186by RSI until 48 h post-inoculation with the peak observed a3tof1125 h postinoculation (Figure 1b), indicating that this CML was upregulated by RSI

Summary

Introduction

Plants are frequently exposed to potentially pathogenic microbes and have evolved a sophisticated defense system initiate defense upon pathogen attack This process is largely regulated at the transcriptional levels by the action of different transcription factors, which themselves are activated by early stages of signaling. The Ca2+ signature is decoded and transmitted downstream by different Ca2+ sensors including calmodulin like (CML) proteins, calcium-dependent protein kinases (CDPKs) and calcineurin B-like proteins (CBLs). These Ca2+ sensors relay or decode the encoded Ca2+ signals into specific cellular and physiological responses in order to survive challenges by pathogens [3,4,5].

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Results

Conclusion