AbstractCochliobolus heterostrophusis a crucial pathogenic fungus that causes southern corn leaf blight (SCLB) in maize worldwide, however, the virulence mechanism of the dominant race O remains unclear. In this report, the single-cell level of pathogen tissue at three infection stages were collected from the host interaction-situ, and were performed next-generation sequencing from the perspectives of mRNA, circular RNA(circRNA) and long noncoding RNA(lncRNA). In the mRNA section, signal transduction, kinase, oxidoreductase, and hydrolase, et al. were significantly related in both differential expression and co-expression between virulence differential race O strains. The expression pattern of the traditional virulence factors nonribosomal peptide synthetases (NPSs), polyketide synthases (PKSs) and small secreted proteins (SSPs) were multifarious. In the noncoding RNA section, a total of 2279 circRNAs and 169 lncRNAs were acquired. Noncoding RNAs exhibited differential expression at three stages. The high virulence strain DY transcribed 450 more circRNAs than low virulence strain WF. Informatics analysis revealed numbers of circRNAs which positively correlate with race O virulence, and a cross-kingdom interaction between the pathogenic circRNA and host miRNA was predicted. An important exon-intron circRNA Che-cirC2410 combines informatics characteristics above, and highly expressed in the DY strain. Che-cirC2410 initiate from the pseudogenechhtt, which doesn’t translate genetic code into protein. In-situ hybridization tells the sub-cellular localization of Che-cirC2410 include pathogen`s mycelium, periplasm, and the diseased host tissues. The target of Che-cirC2410 was predicted to be zma-miR399e-5P, and the interaction between noncoding RNAs was proved. More, the expression of zma-miR399e-5P exhibited a negative correlation to Che-cirC2410 in vivo. The deficiency of Che-circ2410 decreased the race O virulence. The host resistance to SCLB was weakened when zma-miR399e-5P was silenced. Thus, a novel circRNA-type effector and its resistance related miRNA target are proposed cautiously in this report. These findings enriched the pathogen-host dialogue by using noncoding RNAs as language, and revealed a new perspective for understanding the virulence of race O, which may provide valuable strategy of maize breeding for disease resistance.Author SummaryThe southern corn leaf blight (caused byCochliobolus heterostrophus) is not optimistic in Asia, however we have limit knowledge about the infection mechanism of the dominantC.heterostrophusrace O. We take full advantage of the idealC.heterostrophusgenome database, laser capture microdissection and single-cell level RNA sequencing. Hence, we could avert the artificial influence such as medium, and profile the real gene mobilization strategy in the infection. The results of coding RNA section were accessible, virulence related genes (such as the signal transduction, PKS, SSP) were detected in RNA-seq,which accord with previous reports. However, the results of noncoding RNA was astonished, 2279 circular RNAs (circRNA) and 169 long noncoding RNAs (lncRNA) were revealed in our results. Generally, the function of noncoding RNA was hypothesized in single species, but we boldly guess that the function of circRNA is rather complicated in the pathogen-host interaction. Finally, the circRNA in-situ hybridization (ISH) demonstrate the secretion of pathogen circRNA into the host tissue. By bioinformatic prediction, we found a sole microRNA target, and proved the interaction between circRNA and microRNA. These findings are likely to reveal a novel pathogen effector type: secreted circRNA.
Signal Transduction at the Single-Cell Level: Approaches to Study the Dynamic Nature of Signaling Networks