MeCIPK23 interacts with Whirly transcription factors to activate abscisic acid biosynthesis and regulate drought resistance in cassava

As one of the most important food and energy crops in the world, cassava (Manihot esculenta) is high tolerant to drought and poor nutritional environment. However, the key regulators of drought response in cassava remain elusive. In this study, we found that MeWHYs could physically interact with MeCIPK23 in vivo and in vitro, as revealed by yeast two-hybrid, biomolecular fluorescence complementation (BiFC), luciferase (LUC) complementation, and pull-down assays. Moreover, we highlighed their roles in regulating drought response in cassava. Under drought stress conditions, the expression of MeCIPK23 and MeWHYs are up-regulated. In addition, MeCIPK23 interacts with MeWHYs, which directly bind to the PB element in the promoter of MeNCED1 and activate its transcription. Then the up-regulated expression of MeNCED1 results in elevated ABA biosynthesis and enhanced drought stress response. Therefore, this study provides new insight into the drought-resistance mechanism in cassava and potential strategies for further crop breeding and germplasm enhancement.


Dear editor,
With the global climate change, drought has become one of the most serious environmental stresses that affect crop yield (Fahad et al., 2017). As one of the most important food and energy crops, cassava (Manihot esculenta) feeds about 750 million people in the world, especially in Africa . It is widely known that cassava is highly tolerant to drought and poor nutritional environment (De Souza et al., 2017). However, the key regulators of drought response in cassava remain elusive. In our previous study, we have identified Whirly (MeWHY) transcriptional factors and revealed their roles in modulating plant disease resistance against cassava bacteria blight (CBB) through interacting with MeWRKY75 . Herein, we further found that MeWHYs could physically interact with MeCIPK23 (Hu et al., 2015;Yan et al., 2018), as revealed by yeast two-hybrid, biomolecular fluorescence complementation (BiFC), luciferase (LUC) complementation and pull-down assays (Figure 1a-d).
WHY family proteins widely exist in plants and play multiple roles in modulating growth and development Prikryl et al., 2008). In barley, WHY1 regulates drought stressinduced leaf senescence through modulation of the expression of drought stress-related genes and senescence-related genes (Janack et al., 2016). Previous study has identified a total of 25 MeCIPKs and shown that the transcripts of some MeCIPKs including MeCIPK23 could be significantly regulated by drought stress and exogenous abscisic acid (ABA) treatment (Hu et al., 2015). In addition, OsCIPK23 positively regulates plant drought stress resistance (Hu et al., 2015). Based on previous studies (Hu et al., 2015;Janack et al., 2016) Figure 1g). Compared with mock, gene-silenced plants exhibited obvious drought stress sensitivity with more wilted leaves, higher water loss rate and higher electric leakage (EL) upon drought stress treatment for 20 day, and the effects are more obvious in triple-and tetrad-silenced plants (Figure 1h, i, r). More wilted leaves, higher water loss rate and higher EL under drought stress conditions reflected worse leaf phenotype, lower water-holding capacity and severer plasma membrane damage, respectively, suggesting that MeWHY1-, MeWHY2-, MeWHY3and MeCIPK23-silenced plants displayed enhanced drought stress sensitivity in cassava.
Abscisic acid plays a crucial role in plant drought stress resistance (Cai et al., 2017). Therefore, we wondered whether MeWHYs and MeCIPK23 regulated ABA level. We firstly detected the expression of MeNCED genes, which encode the key enzymes controlling ABA biosynthesis (Cai et al., 2017). Because only the transcript of MeNCED1 among six MeNCEDs exhibited a dramatic decrease in MeWHYs-silenced cassava leaves (Figure 1j), this gene was selected for further analysis. Moreover, MeWHYs-and MeCIPK23-silenced cassava leaves had lower expression levels of MeNCED1 after drought stress treated for 20 days in comparison to mock (Figure 1k). Consistent with compromised MeNCED1 expression level, ABA content was also dramatically lower in MeWHYs-and MeCIPK23-silenced cassava leaves (Figure 1l).
Interestingly, we found a WHY-binding PB motif existing in the promoter region of MeNCED1 (Figure 1m), which has previously been suggested as the target of WHY proteins (Desveaux et al., 2005;Liu et al., 2018). Then, we analysed whether MeNCED1 was a direct target of MeWHYs. Firstly, electrophoretic mobility shift assay (EMSA) indicated that MeWHYs could bind to the promoter region (À1312 to À1262) with PB motif of MeNCED1, since a second band with lower gel shift rate appeared and increased with the addition of MeWHY proteins (Figure 1m). However, MeWHYs could not bind to the mutated probe with mutated PB motif (Figure 1m), confirming that MeWHYs could specifically bind to the PB motif. In addition, ChIP-PCR suggested that the promoter region of MeNCED1 with PB motif was largely enriched by MeWHYs, and the enrichment levels were higher in MeCIPK23 overexpressing background but lower in MeCIPK23-VIGS background, indicating that MeCIPK23 could positively regulate the ability of MeWHYs to bind to PB motif (Figure 1n).
Moreover, three MeWHYs could significantly activate the activity of MeNCED1 promoter in dual LUC reporter system (Figure 1o). To sum up, these results suggested that MeNCED1 is a direct target of MeWHYs. Notably, MeCIPK23 overexpression could enhance the activity of MeNCED1 promoter and enhance the effects of MeWHYs on activating the activity of MeNCED1 promoter under mock conditions, but the effects of MeCIPK23 overexpression were significantly lower under MeWHY1/2/3-VIGS background (Figure 1o), indicating that the interaction between MeCIPK23 and MeWHYs could direct regulate the activity of increase of water loss rate and relative EL was dramatically compromised by ABA treatment in MeWHYs-and MeCIPK23silenced cassava leaves (Figure 1s-t), indicating that ABA biosynthesis is directly involved in MeWHYs-and MeCIPK23-mediated drought stress resistance in cassava.
Taken together, we proposed a potential model for MeCIPK23-MeWHYs-mediated drought stress response in cassava (Figure 1u). Under drought stress conditions, the expression of MeCIPK23 and MeWHYs are up-regulated. In addition, MeCIPK23 interacts with MeWHYs, which directly bind to the PB element in the promoter of MeNCED1 and activate its transcription. Then, the up-regulated expression of MeNCED1 results in elevated ABA biosynthesis and enhanced drought stress response. Therefore, this study provides new insight into the drought-resistance mechanism in cassava and potential strategies for further crop breeding and germplasm enhancement.