January 2023
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93 Reads
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11 Citations
The HSC70/HSP70 family of heat shock proteins are evolutionarily conserved chaperones involved in protein folding, protein transport, and RNA binding. Arabidopsis HSC70 chaperones are thought to act as housekeeping chaperones and as such are involved in many growth‐related pathways. Whether Arabidopsis HSC70 binds RNA and whether this interaction is functional has remained an open question. We provide evidence that the HSC70.1 chaperone binds its own mRNA via its C‐terminal short variable region (SVR) and inhibits its own translation. The SVR encoding mRNA region is necessary for HSC70.1 transcript mobility to distant tissues and that HSC70.1 transcript and not protein mobility is required to rescue root growth and flowering time of hsc70 mutants. We propose that this negative protein‐transcript feedback loop may establish an on‐demand chaperone pool that allows for a rapid response to stress. In summary, our data suggest that the Arabidopsis HSC70.1 chaperone can form a complex with its own transcript to regulate its translation and that both protein and transcript can act in a noncell‐autonomous manner, potentially maintaining chaperone homeostasis between tissues.
![Cas9- and gNIA1-TLS fusion constructs are functional and mobile in adult plants
a, Appearance of grafted plants (43 days after grafting) grown on soil and treated with 5 μM estradiol. b, RT–PCR detection (45 PCR cycles) of Cas9 and gNIA1 transcripts in rootstock (R) and grafted wild-type tissues samples from silique (Sil), flower (Flo), cauline leaf (Cau), stem (St), and rosette (Ro). Four independent replicates (each sample a pool of three grafted plants) were analyzed. Stars indicate RT–PCR Cas9-TLS and gNIA1-TLS amplicons detected in wild-type scion samples. Kan and Hyg transcripts serve as contamination controls. Note that transcript absence was confirmed by 50 PCR cycles. c, RT–qPCR detection of mobile Cas9-TLS transcripts in scion tissues and in grafted rootstock (Root). y-axis, mean 2−ΔΔct\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$2^{-\Delta\Delta c_t}$$\end{document} values; log10 scale. Each value represents the mean of three independent biological replicates presented as black dots on the bar. Note that every 2−ΔΔct\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$2^{-\Delta\Delta c_t}$$\end{document} data point below the dashed line represents technical background as measured with wild-type scion samples grafted onto wild-type rootstocks with no Cas9 transcript present. Significance was calculated using Student’s t-test (two tails); P values indicated by a, b, and c: a,b = 8.31567 × 10⁻²²; b,c = 1.16396 × 10⁻²²; a,c = 1.90267 × 10⁻¹¹. d, Genomic PCR to detect NIA1 edited fragments in siliques and flowers. Samples were from the same plant material as analyzed in c. e, Mobile CRISPR–Cas9-induced genome edits confirmed by Sanger sequencing.
Source data](https://www.researchgate.net/publication/366817334/figure/fig3/AS:11431281174707681@1689304001881/Cas9-and-gNIA1-TLS-fusion-constructs-are-functional-and-mobile-in-adult-plants-a_Q320.jpg)
![Cas9- and gNIA1-TLS2 fusion constructs are mobile and functional in B. rapa/Arabidopsis grafted plants
a, Appearance of grafted plants (20 and 40 days after grafting) grown on MS medium and treated with 5 μM estradiol. Red arrows indicate the graft junction. b, RT–PCR detection (45 PCR cycles) of Cas9 and gNIA1 transcripts in rootstock, and grafted wild-type tissues samples from silique, flower, leaf, and stem from one independent replicate (the other three replicates are presented in Extended Data Fig. 3) was analyzed. Stars, RT–PCR Cas9-TLS and gNIA1-TLS amplicons detected in B. rapa wild-type scion samples. Kan and Hyg transcripts serve as contamination controls. Note that transcript absence was confirmed by 50 PCR cycles. c, RT–qPCR detection of mobile Cas9-TLS transcripts in scion tissues and in grafted rootstock (Root). y-axis, mean 2−ΔΔct\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$2^{-\Delta\Delta c_t}$$\end{document} values; log10 scale. Each value represents the mean of three independent biological replicates presented as black dots on the bar. Note that every 2−ΔΔct\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$2^{-\Delta\Delta c_t}$$\end{document} value below the dashed line represents technical background as measured with wild-type scion samples grafted onto wild-type rootstocks with no Cas9 transcript present. Significance was calculated using Student’s t-test (two tails); P values indicated by a, b and c: a,b = 3.48073 × 10⁻⁸; b,c = 2.29801 × 10⁻⁷; a,c = 3.43932 × 10⁻⁸. d, Genomic PCR to detect NIA1 edited fragments in siliques and flowers. Samples were from the same plant material as analyzed in b,c. e, Mobile CRISPR–Cas9-induced genome edits confirmed by Sanger sequencing.
Source data](https://www.researchgate.net/publication/366817334/figure/fig5/AS:11431281174723217@1689304004405/Cas9-and-gNIA1-TLS2-fusion-constructs-are-mobile-and-functional-in-B-rapa-Arabidopsis_Q320.jpg)