Homeobox 27, a Homeodomain Transcription Factor, Confers Tolerances to CMV by Associating with Cucumber Mosaic Virus 2b Protein

Abstract

Transcription factors (TFs) play an important role in plant development; however, their role during viral infection largely remains unknown. The present study was designed to uncover the role transcription factors play in Cucumber mosaic virus (CMV) infection. During the screening of an Arabidopsis thaliana (Col-0) transcription factor library, using the CMV 2b protein as bait in the yeast two-hybrid system, the 2b protein interacted with Homeobox protein 27 (HB27). HB27 belongs to the zinc finger homeodomain family and is known to have a regulatory role in flower development, and responses to biotic and abiotic stress. The interaction between CMV 2b and HB27 proteins was further validated using in planta (bimolecular fluorescence complementation assay) and in vitro far-Western blotting (FWB) methods. In the bimolecular fluorescence complementation assay, these proteins reconstituted YFP fluorescence in the nucleus and the cytoplasmic region as small fluorescent dots. In FWB, positive interaction was detected using bait anti-MYC antibody on the target HB27-HA protein. During CMV infection, upregulation (~3-fold) of the HB27 transcript was observed at 14 days post-infection (dpi) in A. thaliana plants, and expression declined to the same as healthy plants at 21 dpi. To understand the role of the HB27 protein during CMV infection, virus accumulation was determined in HB27-overexpressing (HB27 OE) and knockout mutants. In HB27-overexpressing lines, infected plants developed mild symptoms, accumulating a lower virus titer at 21 dpi compared to wild-type plants. Additionally, knockout HB27 mutants had more severe symptoms and a higher viral accumulation than wild-type plants. These results indicate that HB27 plays an important role in the regulation of plant defense against plant virus infection.

Investigating the interactions of the cucumber mosaic virus 2b protein with the viral 1a replicase component and the cellular RNA silencing factor Argonaute 1

“…For example, a key feature of intrinsically disordered proteins is their ability to interact with multiple partners (Okoye et al, 2022;Sipko et al, 2024). This multivalency could explain variously reported interactions (in addition to those explored in this paper) with: DNA (Ham et al, 1999;Sueda et al, 2010), a catalase (Inaba et al, 2011) and a calmodulin-like factor in tobacco (Nakahara et al, 2012), cucumber RNA-directed RNA polymerase 1 (Kumari et al, 2021), an A. thaliana zinc finger protein HB27 (Rattan et al, 2022), and A. thaliana jasmonate ZIM-domain (JAZ) proteins 1, 3, 6 and 10 ( Wu et al, 2017). Intrinsically disordered proteins, including at least one plant viral protein (the p26 movement protein of pea enation mosaic virus 2), can phase separate and enter membrane-free organelles composed of protein-rich droplets (Brangwynne et al, 2009;Brady et al, 2017;Mitrea and Kriwacki, 2016;Wallmann and Kesten, 2020;Brown et al, 2021).…”

Investigating the Interactions of the Cucumber Mosaic Virus 2b Protein with the Viral 1a Replicase Component and the Cellular RNA Silencing Factor Argonaute 1

“…For example, a key feature of intrinsically disordered proteins is their ability to interact with multiple partners (Okoye et al, 2022;Sipko et al, 2024). This multivalency could explain variously reported interactions (in addition to those explored in this paper) with: DNA (Ham et al, 1999;Sueda et al, 2010), a catalase (Inaba et al, 2011) and a calmodulin-like factor in tobacco (Nakahara et al, 2012), cucumber RNA-directed RNA polymerase 1 (Kumari et al, 2021), an A. thaliana zinc finger protein HB27 (Ra an et al, 2022), and A. thaliana jasmonate ZIM-domain (JAZ) proteins 1, 3, 6 and 10 (Wu et al, 2017). Intrinsically disordered proteins, including at least one plant viral protein (the p26 movement protein of pea enation mosaic virus 2), can phase separate and enter membrane-free organelles composed of protein-rich droplets (Brangwynne et al, 2009;Brady et al, 2017;Mitrea and Kriwacki, 2016;Wallmann and Kesten, 2020;Brown et al, 2021).…”

Investigating the Interactions of the Cucumber Mosaic Virus 2b Protein with the Viral 1a Replicase Component and the Cellular RNA Silencing Factor Argonaute 1

“…For example, a key feature of intrinsically disordered proteins is their ability to interact with multiple partners [45,69]. This multivalency could explain variously reported interactions (in addition to those explored in this paper) with: DNA [24,70], a catalase [71] and a calmodulin-like factor in tobacco [72], cucumber RNA-directed RNA polymerase 1 [73], an A. thaliana zinc finger protein HB27 [74], and A. thaliana jasmonate ZIM-domain (JAZ) proteins 1, 3, 6, and 10 [75]. Intrinsically disordered proteins, including at least one plant viral protein (the p26 movement protein of pea enation mosaic virus 2), can phase separate and enter membrane-free organelles composed of protein-rich droplets [46,[76][77][78][79].…”