The Exserohilum turcicum effector EtEC81 reprograms alternative splicing in maize and activates immunity [RIP-Seq]

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 events 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. The RIP assay was performed as previously described with minor modification65-67. Briefly, total RNA was extracted from maize plants for incubation with MBP-EtEC81 and MBP, respectively. MBP-EtEC81 and MBP were purified with amylose resins (NEB, E8201) washed six times with MBP lysis and washing buffer (150 mM NaCl, 50 mM Tris-HCl pH 7.5, 1 mM EDTA, 1 mM PMSF, 20 units/mL RNase inhibitor [Promega, N251A]). A total of 10 μg RNA and 10 μg purified protein were mixed and treated with 0.5% fromaldehyde for 30 min for cross-linking, following with 125 mM glycine for 5 min to stop the reaction. Add up to 1 mL MBP lysis and washing buffer, and saved 100 μL mixture as input. Then the remaining RNA-protein solution was incubated with 50 μL amylose resins overnight at 4℃ with rotation. The RNA/MBP and RNA/MBP-EtEC81 resin complexes were thoroughly washed six times with MBP lysis and washing buffer. The immunoprecipitated RNAs and input-RNA were extracted with TRIzol reagent (Invitrogen, 15596018). Library construction and sequencing were performed by Novogene Inc (Beijing, China). The library was sequenced on an illumina Novaseq platform (HiSeq-PE150). Raw reads were firstly subjected to quality control using FastQC (v.0.20.1). The filtered reads were mapped to the Zea mays reference genome (https://download.maizegdb.org/Zm-B73-REFERENCE-GRAMENE-4.0/Zm-B73-REFERENCE-GRAMENE-4.0.fa.gz) using BWA mem (v0.7.12). The MACS2 (version 2.1.0) peak calling software to identify regions of IP enrichment over background. The RIP target were identified based on the default parameters (P-value<0.05, and Fold change>2).