Single-cell level transcriptome of the maize pathogenic fungi cochliobolus heterostrophus race O in infection reveal the virulence related genes, and potential circRNA effector

Cochliobolus heterostrophus is 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 pseudogene chhtt, 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 Summary The southern corn leaf blight (caused by Cochliobolus heterostrophus) is not optimistic in Asia, however we have limit knowledge about the infection mechanism of the dominant C.heterostrophus race O. We take full advantage of the ideal C.heterostrophus genome 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.

Annotation with molecular function(MF) was used for analysing the differential 195 which intersect in multiple database, which tell the complication of virulence related 196 gene function (Fig. 3B). between 222-322bp, and 137 circRNAs` length were longer than 3000bp (Fig. 6B).

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Between two strains, DY transcribed around 200 more circRNAs than WF at 233 each stage. 900, 657 and 307 differential circRNAs were revealed at the CO, SM and 234 DM stage separately (P < 0.05) (Fig. 3). Similar to the mRNA transcription feature, 235 the number of circRNAs decrease progressively, while the differential expression 236 level expand along with the infection process (Fig. 3). In GO terms which circRNAs 237 aligned to, several GO terms showed high frequency, for example: (1) ATP binding   268 the host zma-miR399e-5P (Fig 8, Fig 10A, Fig. 10B), and zma-miR399e-5P was 269 predicted to target 35 maize transcripts (Fig. 8), and diverse significant genes, such as 270 serine/threonine kinases, RING finger were found among these targets (Table 2).
271 However, some interaction matrices are complicated: two pathogen circRNAs 272 (circ3462, circ3475) were predicted the interaction with four host miRNA, and the 273 excess targets of miRNA beyond our cognitive (S1_dataset, page1).
320 Considerable zma-miR399e5P was detected in the cytoplasm of the mesophyll cells 321 but emerged rarely in vascular cells (S3 Fig.), when the leaf was infected with the 322 pathogen, the density of the zma-miR399e-5P signal declined in mesophyll cells (Fig.   323 14).

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To further determine the sources of the two noncoding RNAs, we tested the pure 325 plant and fungal colony. The zma-miR399e-5P were detected in maize only, while 326 Che-cirC2410 was detected in the fungal pathogen only (S3 Fig., S4 Fig.).

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The ISH results provide the probability of interface between Che-circ2410 and 328 zma-miR399e5P, additionally, a dual-luciferase reporter system was used to test this 329 (Fig. 15). The fluorescence intensity of the negative control was 10.266, and that of 330 the experimental group, Che-cirC2410-zma-miR399e5P, was 48% of the negative 331 control (4.907, P = 0.0012) (S4_Table). The results indicated a significant variation 332 resulting from the sponge. Moreover, to exclude the false positive that the reduction 333 of fluorescence intensity generated from a potential plasmid sequence, we introduced 334 a nucleotide mutation of the Che-cirC2410, and the fluorescence intensity was 335 recovered to 95% of the negative control (Fig. 15).

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The interaction between noncoding RNAs was demonstrated in vitro, more, the 337 expression of zma-miR399e5P was evaluated in vivo. When the Che-cirC2410 of C.

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In soybean, it was reported that nine miRNAs were regulated by noncoding

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Interactions between circRNA and maize miRNA and mRNA were predicted 500 using the psRNATarget software (http://plantgrn.noble.org/psRNATarget/)(100). The  The circRNA treatment by RNase R was performed as described by 524 Ashwal(104) with modifications; the total quantity for digestion was 10 μg, and the The zma-miR399e5P mimics sequence is GGGCUUCUCUUUCUUGGCAGG; 555 and the antagomir sequence is CCUGCCAAGAAAGAGAAGCCC. All mimics and 556 antagomir were chemically modified by methoxy group, which were finished by 557 Shanghai Sangon Biotech. The mimics and antagomir water solution (Rnase free, 1 558 nmol/mL) were injected by needleless injector at the separate sides of vein, after this, 559 the pathogen were inoculated as previously described.

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ATMT was performed to knock out the chhtt gene, which transcribes Che-