The Exserohilum turcicum effector EtEC81 reprograms alternative splicing in maize and activates immunity
Some pathogen-derived effectors reprogram mRNA splicing in their host plant to regulate plant immune responses. The fungus Exserohilum turcicum is the causal agent of northern corn leaf blight, a damaging maize (Zea mays) disease. However, the low efficiency of genetic transformation of E. turcicum has hampered research on its effectors and whether E. turcicum effectors interfere with RNA splicing remained unknown. Here, using an alternative splicing (AS) reporter system, we identified the secreted protein EtEC81 (Exserohilum turcicum effector 81), which modulates the AS of maize pre-mRNAs and negatively regulates the pathogenicity of E. turcicum. EtEC81 physically interacts with EtEC81-interactiNG protein 1 (ZmEIP1), which associates with maize spliceosome components, regulating AS and positively regulating the defense response against E. turcicum. EtEC81 binding further enhanced the effect of ZmEIP1 on AS. Transcriptome analysis revealed 119 common genes with altered AS in maize plants transiently overexpressing ZmEIP1 or EtEC81, suggesting that these factors cause the mis-regulation of cellular activities and thus induce immune responses. We used RT-qPCR to verify representative AS events in the plants transiently overexpressing ZmEIP1 and EtEC81. Together, our results suggest that the EtEC81 effector targets ZmEIP1 to reprogram pre-mRNA splicing in maize. Overall design: The fourth leaves of transgenic maize plants with virus-induced overexpression of EtEC81 or GFP and T2 transgenic maize plants overexpressing ZmEIP1 and WT plants were collected for total RNA extraction and RNA-seq library construction. The RNA-seq libraries were sequenced on an Illumina HiSeq platform in paired-end mode with a read length of 150 bp (Novogene, Beijing, China).  The raw reads were filtered using ht2-filter in the HTQC package (1.92.1) with default parameters to remove low-quality reads. The filtered reads were compared to the reference maize genome (Zea mays RefGen_V4) using TopHat (2.0.11) to remove host genome sequences. The mapped reads were assembled, and fragments per kilobase of transcript per million mapped reads (FPKM) values were calculated using Cufflinks. The Cufflinks output GTF file and the TopHat output BAM file were loaded into rMATS.3.2.2 to identify differential AS events. AS events with a P value < 0.05 were considered to be differential AS events.