In plant-pathogen co-evolution, plants employ two-layered immunity to counterattack the invasion of pathogens, namely pathogen/microbe-associated molecular pattern- (PAMP/MAMP-) triggered immunity (PTI) and effector-triggered immunity (ETI) (Jones and Dangl, 2006). PTI is the first layer of plant immunity activated by the recognition of the PAMPs/MAMPs and pattern recognition receptors (PRRs), such as bacterium-derived flg22 and fungus-derived chitin, to effectively protect plants from the invasion of potential pathogens (Boller and Felix, 2009). The typical PTI responses include the activities of MAPK cascades, the influx of [Ca2+]cyt, the burst of reactive oxygen species (ROS), the induction of basal defense-related genes and the callose deposition at the infected sites, and so on (Boller and Felix, 2009). However, adapted pathogens can subvert PTI by delivering effectors in host cells (Dou and Zhou, 2012). In turn, plants have involved resistance (R) proteins to recognize these specific effectors resulting in ETI, which offers strong resistance and is often associated with the hypersensitive response (HR) (Cui et al, 2015).
MiRNAs are a category of 20-24-nucleotide (nt) non-coding RNAs expressed from MIR genes that regulate target gene expression by sequence-complementary DNA methylation or mRNA cleavage, or translational inhibition (Yu et al, 2017). Based on their roles in the regulation of gene expression, miRNAs act as fine-tuners of various biological processes controlling growth and stress-induced responses (Tang and Chu, 2017). Growing evidence shows that microRNAs (miRNAs) are involved in plant immunity (Padmanabhan et al, 2009, Katiyar-Agarwal and Jin, 2010, Baldrich and San Segundo, 2016, Huang et al, 2016, Tang and Chu, 2017). In Arabidopsis, the PAMP molecule flg22 induces the expression of miR160a and miR393, whereas suppresses the accumulation of other nine miRNAs following the inoculation of the virulent Pseudomonas syringae pv. tomato DC3000 (Pst DC3000) (Navarro et al, 2006, Li et al, 2010). miRNAs are also involved in plant ETI. In Arabidopsis, the amounts of miR863-3p are increased during ETI triggered by Pseudomonas syringae carrying effector avrRpt2 (Pst DC3000(avrRpt2)). miR863-3p fine-tunes the amplitude and timing of defense responses by suppressing the target genes that play reverse functions in rice immunity. At the earlier infection stage, miR863-3p promotes immunity by suppressing the expression of typical receptor-like pseudokinase1 (ARLPK1) and ARLPK2, which negatively regulate plant defense (Niu et al, 2016). At a later infection stage, miR863-3p limits immunity amplitude by silencing SERRATE, which is required for miRNA accumulation and positively regulates plant defense (Niu et al, 2016).
Rice blast disease caused by Magnaporthe oryzae (M. oryzae) ranks the first fungal disease threatening food production worldwide. The utilization of disease resistance genes in cultivars generates an economically and environment-friendly strategy for disease control. Intriguingly, miRNAs play important roles in rice resistance against M. oryzae (Li et al, 2014, Li et al, 2016). Nowadays, more than 15 miRNAs have been characterized as the regulators of rice blast disease resistance. miR159(Chen et al, 2021), miR160 (Li et al, 2014), miR162 (Salvador-Guirao et al, 2019, Li et al, 2020), miR166 (Salvador-Guirao et al, 2018), miR398(Li et al, 2019), miR7695(Campo et al, 2013), and miR812w (Campo et al, 2021) positively regulate rice resistance against M. oryzae, whereas miR156 (Zhang et al, 2020), miR164 (Wang et al, 2018b), miR167(Zhao et al, 2019b), miR169 (Li et al, 2017), miR319 (Zhang et al, 2018), miR396 (Chandran et al, 2019), miR439 (Lu et al, 2021), miR444b.2 (Xiao et al, 2017), and miR1873 (Zhou et al, 2020) negatively regulate rice disease resistance. Among these miRNAs, some and their target genes are involved in both rice immunity and growth. For example, miR162 balances immunity and grain yield via Dicer-like 1 (DCL1). Overexpression of miR162 enhances rice blast resistance whereas compromises yield accompanied by the suppressed expression of DCL1; in contrast, blocking miR162a improves yield whereas penalizes immunity associated with enhanced expression of DCL1 (Salvador-Guirao et al, 2019, Li et al, 2020).
miR1432 is a conserved miRNA family in plants involving in development and defense responses against biotic or abiotic stresses. In barley, the amounts of miR1432-5p increase during barley development (Pacak et al, 2016). In maize, miR1432 is down-regulated in meristem under chilling stress (Aydinoglu, 2020). In wheat, miR1432 in leaves is down-regulated by water deficit in presence of mycorrhizal treatment (Fileccia et al, 2019). In wild emmer wheat (Triticum turgidum ssp. dicoccoides), the expression of miR1432 is induced in root under drought stress (Kantar et al, 2011). In rice, miR1432 is predicted as a key regulator for rice grain-filling and targets Acyl-CoA thioesterase (OsACOT) (Hu et al, 2018). Blocking miR1432 significantly enhances grain weight resulting in overall grain yield up more than 17% in a field trial via expressing a Short Tandem Target Mimic of miR1432 (STTM1432). Moreover, overexpression of OsACOT resembled the yield traits of STTM1432 plants (Zhao et al, 2019a), indicating miR1432 is involved in grain-filling via OsACOT. The expression of rice miR1432 is also responsive to the infection of M. oryzae (Li et al. 2014). However, it remains largely unknown how miR1432 coordinates rice immunity and yield traits.
In this study, we constructed the transgenic lines overexpressing miR1432, the lines blocking miR1432 by expressing a target mimic of miR1432, and the lines overexpressing the target genes of miR1432, OsACOT, and OsEFH1 (EF-hand family protein 1), respectively. We explored the blast disease resistance and yield traits of these lines. We found that miR1432 negatively regulates rice immunity and yield, whereas blocking miR1432 leads to enhanced blast disease resistance and increased yield. We revealed that OsEFH1 was targeted by miR1432 and acted as a positive regulator of rice blast disease resistance but a negative regulator of rice yield. Further study revealed that the miR1432-OsEFH1 module regulated rice PTI responses, whereas OsACOT had no obvious effect on rice immunity. Altogether, our results revealed that a miRNA coordinates rice yield and immunity via different target genes that play differential roles, and indicated the capacity of the miR1432-targets module in the improvement of both immunity and yield in rice.