title: The role of cauliflower mosaic virus P6 in modulating plant hormone signalling
Creators: name: Mater, Yousef Jalil
Publisher: name: University of Glasgow
Year: 2021

description: Pathogens have evolved mechanisms to manipulate host responses and physiology to facilitate their survival, spread, and promote invasion. The production of effector proteins by pathogens to manipulate and control host cellular mechanisms is a key feature of host-pathogen interactions and is employed by diverse types of pathogens, from fungi and bacteria to viruses. Cauliflower mosaic virus (CaMV) a plant pararetrovirus has been shown to modulate phytohormone synthesis and signalling pathways in infected plants. These include those involving salicylic acid (SA) signalling pathway, auxin (Aux) signalling pathway, jasmonic acid (JA) signalling pathway. The modulation of phytohormone signalling can be enacted directly or indirectly, impacting diverse aspects of plant physiology, including growth, development, reproduction, and responses to various biotic and abiotic stresses. CaMV encodes a multifunctional 520 amino acid (aa) protein, P6, which was initially identified as playing an essential role in virus replication by facilitating the expression of multiple open reading frames from the virus-encoded 35S RNA transcript. More recently P6 has been shown to function as an effector protein, where Arabidopsis plants ectopically expressing P6 exhibit symptom-like phenotypes (stunting and yellowing) and alterations in plant defense signalling. In particular, P6 suppresses innate immunity by down regulating aspects of responses to salicylic acid (SA) and enhancing jasmonic acid/ethylene (JA/ET) responses. This result in increased susceptibility to biotrophic pathogens but increased resistance to herbivores and necrotrophic pathogens. The translational transactivation function and the suppression of innate immunity are both dependent on the ability of P6 to bind to the multifunctional receptor kinase TOR which plays a key role in multiple aspects of signalling and biochemical responses in eukaryotic organisms including plants. However, the role of P6-TOR interaction in the ability of P6 to modulate other aspects of hormone signalling in plants in particular those involving JA, ET, and Aux are unknown. To dissect the role of the interaction of P6 with TOR, independent homozygous transgenic Arabidopsis plants were constructed that express either a wild-type P6 or a mutant version of P6 with a deletion of the TOR binding domain (Δaa 136–182), P6(∆TOR) were constructed and analyzed at the transcriptional and phenotypic level. Transgenic Arabidopsis lines in Arabidopsis Col-0 background expressing P6 wildtype sequence (P6WT) fused to a C-terminal GFP, and P6 (∆TOR) fused to a C-terminal GFP were generated under the control of a constitutive promoter (35S), pEZR-P6WTGFP and pEZR-P6(∆TOR)-GFP lines, or under the control of a β-estradiol-inducible promoter; pER8-P6WT-GFP and pER8-P6(∆TOR)-GFP lines and two lines of each newly constructed transgenic Arabidopsis line were selected for further analysis. Our western blot and confocal analysis indicated that all pER8 lines had a higher level of transgene expression than pEZR lines and had a controllable expression to avoid unwanted phenotypes. Transgenic Arabidopsis lines expressing (P6WT) and P6(∆TOR) were used to highlight the expression impact on the phytohormonal signalling pathways and responses. The work described here was performed to confirm that P6 modulates phytohormonal signalling pathways, particularly the P6 impact on the ethylene signalling pathway and auxin signalling pathway. Transgenic Arabidopsis lines expressing P6 and P6(∆TOR) were phenotypically assessed using ethylene sensitivity assay and auxin transport inhibitor treatment. The transgenic Arabidopsis lines expressing P6 showed a decrease in ethylene sensitivity compared to Arabidopsis Col-0. Transgenic Arabidopsis lines expressing P6(∆TOR) retained wild-type sensitivity to ethylene, i.e., P6 modulates the ethylene signalling pathway in a TOR binding domain manner. Both transgenic Arabidopsis lines expressing P6 and P6(∆TOR) displayed an increase in the resistance to an auxin transport inhibitor compared to Arabidopsis Col-0, i.e., P6 does not require the presence of a functional TOR binding domain to modulate the auxin signalling pathway. Taken together, these data suggest that P6 modulates both auxin and ethylene signalling pathways during CaMV infection in nature. P6, via its TOR-binding domain, can modulate ethylene signalling pathways by interacting with the plant TOR kinase, but in the case of auxin signalling interactions, another domain of P6 may be important. Here, the regulatory impact of P6 on plant gene expression profiles were investigated by executing RNA sequencing (RNA-seq). The transgenic line expressing P6 from an estradiol inducible promoter exhibited an expression profile distinct from the P6(∆TOR) expressing line. They displayed a different impact on the global Arabidopsis gene expression profile, including genes known to participate in plant development and biotic and abiotic stress responses, i.e., genes contribute to defense response, stress response, signalling, metabolism, cell proliferation, and differentiation. Our results reveal P6 expression leads to extensive regulation of expression of genes frequently used as a marker for the plant defense and phytohormones signalling pathway, including SA, JA/ET, Aux, abscisic acid (ABA), brassinosteroids (BR), and gibberellins (GA) pathway. Moreover, P6 suppressed the expression of several genes associated with Plant responses more than P6(∆TOR), i.e., the lack of TOR binding domain would attenuate the P6 ability to suppress the expression of several important genes, resulting in modification of the P6 impact on the plant phenotypes and responses. Interestingly, some genes are regulated by different P6 domains, i.e., in a non-TOR binding domain manner. Collectively, these findings highlight both the P6 impact and the TOR binding domain's robust role in modulating Arabidopsis responses.