Frontline Warrior microRNA167: A Battle of Survival

Abstract

Plant pathogens such as viruses are detrimental to the survivorship of plant species. Coinfection of maize chlorotic mottle virus (MCMV) and the sugarcane mosaic virus (SCMV) causes a deadly disease in maize. An investigation by Liu et al. (2022) showed the role of Zma-miR167 in positively imparting resistance against the MCMV and SCMV. The authors identified ZmARF3 and ZmARF30 as the targets of Zma-miR167. ZmARF3 and ZmARF30 were identified as transcription factors that bind the cis-element in ZmPAO1 promoters to activate its expression. The authors showed how the Zma-miR167-ZmARF3/30-ZmPAO1 module functions differently in resistant and susceptible lines with high expression of Zma-miR167 in resistant lines correlated with the resistant phenotype. Finally, the authors concluded that MCMV-encoded p31 protein enhances ZmPAO1 enzyme activity for its survival in the host.

Maize is considered a major staple food crop vulnerable to disease outbreaks. Maize Lethal Necrosis (MLN) is one of the severe diseases caused by coinfection of the maize chlorotic mottle virus (MCMV) and the sugarcane mosaic virus (SCMV) [1]. Potyviruses, including SCMV, are diverse and ubiquitous in nature, unlike MCMV; hence, MLN disease outbreaks depend on the emergence of MCMV [1,2]. Until now, no maize varieties have complete resistance to MCMV [3,4] and the molecular mechanism of MCMV infection remains unexplored. Recent MLN outbreaks have become a significant concern for maize production and global food security [5].

How do the host and pathogen help each other to evolve? Pathogen infection is critical for any host to increase fitness and sustain itself in an unfavorable environment. Similarly, pathogens exploit the host machinery to thrive and escape the host defense response. Liu et al. (2022) investigated the role of maize Zma-miR167-ARF3/30-mediated resistance against MCMV and the use of ZmPAO1 as a counter-defense response employed by MCMV [6]. MicroRNAs are post-transcriptional regulators of gene silencing [7]. They are reported to regulate plant growth and development processes as well as biotic and abiotic stress responses [8,9]. Authors discovered that Zma-miR167 was substantially upregulated upon MCMV infection. Overexpression of pre-MIR167b (Zma-miR167OE) decreased MCMV genomic RNA and coat protein (CP) levels and showed mild mosaic symptoms in plants compared to chlorotic and mottle symptoms in wild-type plants upon viral infection. At the same time, Zma-miR167 silencing resulted in severe leaf chlorosis and an increased MCMV genomic RNA and CP accumulation. Moreover, the basal level of Zma-miR167 was examined in resistant and susceptible lines upon MCMV infection, and higher expression of Zma-miR167 in resistant lines suggested that Zma-miR167 may impart resistance against MCMV. Further, the author observed lower coinfection efficiency of MCMV and SCMV in resistant lines than in susceptible lines. These results, in conjunction, indicated that increased expression of Zma-miR167 plays an essential role in providing resistance to MLN.

What is the underlying mechanism of Zma-miR167-mediated resistance to MCMV? To address this question, the authors used an array of experiments for identifying Zma-miR167 target candidates, ZmARF3 and ZmARF30. Increased expression of ZmARF3 and ZmARF30 was observed in Zma-miR167 knockdown, while ZmARF3 and ZmARF30 expression was decreased in Zma-miR167 overexpression lines. Further, Nicotiana benthamiana leaves expressing the GFP fusion of these two target candidates showed reduced green fluorescence and corresponding protein levels upon an increase in Zma-miR167 levels. The virus-induced gene silencing of ZmARF3 and ZmARF30 led to a similar phenotype to Zma-miR167 overexpression, i.e., mild symptoms and reduced genomic RNA and CP levels upon MCMV infection. The findings indicated that Zma-miR167 targets ZmARF3 and ZmARF30, and ZmARF3/30 negatively regulates plant resistance against MCMV.

To understand further the Zma-miR167-ZmARF-mediated signaling cascade, the authors performed transcriptomic analysis of Zma-miR167 overexpression and knockdown lines and their respective controls. The antagonistically expressing common DEGs were evaluated for downstream analysis, including GO enrichment, which revealed that these DEGs primarily regulate the oxidation–reduction biological process. The authors have further performed a promoter scan on DEGs to determine whether these DEGs are also directly regulated by ARF. They narrowed it down to seven DEGs, particularly polyamine oxidase 1 (ZmPAO1), the hydrogen peroxide (H₂O₂) production gene that has binding sites for ZmARFs and participates in the oxidation–reduction process.

To understand the role of ZmPAO1 in the Zma-miR167-ZmARF signaling cascade during MCMV infection, the authors performed various promoter binding and transcriptional activity assays to validate the strong affinity between ZmARF3/ZmARF30 and ZmPAO1 promoter. ZmPAO1 followed the same expression pattern trend as ZmARF3/ZmARF30 in overexpression and knockdown lines of Zma-miR167, and silenced plants of ZmPAO1 are also less sensitive to MCMV infection. Moreover, to explore the correlation between ZmPAO activity and MCMV infection, the authors treated maize plants with the ZmPAO activity inhibitor, Guazatine. The treated plants showed fewer MCMV-CP than the control plants. These findings suggest that the Zma-miR167–ZmARF3/30 module minimizes MCMV infection, whereas ZmPAO1 helps in viral infection.

Intriguingly, although the authors found less MCMV infection in Zma-miR167OE compared to the control, MCMV can still survive in Zma-miR167OE, indicating that MCMV employs a counter-defense mechanism. To understand the MCMV-mediated defense mechanism, the authors performed the yeast two-hybrid interaction between the ZmPAO1 and each of seven MCMV-encoded proteins. The screening revealed an interaction between ZmPAO1 and MCMV-encoded p31 protein. Additional in vivo and in vitro interaction techniques validated the physical interaction between ZmPAO1 and MCMV p31. Moreover, co-expression of p31 with ZmPAO1 enhanced ZmPAO1 activity and hence increased H₂O₂ production compared to the control, indicating p31 is positively maintaining the stability of ZmPAO1. Overall findings suggested Zma-miR167 plays a vital role in resisting the MCMV infection, and MCMV reiterates back involving a counter-defensive approach by enhancing PAO activity (Figure 1). This study provided a promising candidate, Zma-miR167, that can be used to screen the MLN/MCMV resistant varieties. Also, the Zma-miR167-ZmARF3/30-ZmPAO1 module can be utilized in genetic engineering and breeding programs to generate virus-resistant maize plants. Future research may seek to understand the molecular mechanism behind Zma-miR167–ZmARF3/30-mediated regulation of the jasmonic acid, salicylic acid and brassinosteroid signaling pathway to generate MCMV-resistant lines.