Dichlorvos, is a broad-spectrum organophosphorus pesticide that is widely applied in the agricultural industry and considered a pollutant to fish and bees. T. atroviride strain T23, an efficient DDVP-degrading strain, could convert DDVP to dichloroacetic acid, 2,2-dichloroethanol and phosphoric acid through mineralization. RT-qPCR analysis showed TaPon1 -like encoding an organophosphorus hydrolase, is continuously highly expressed in the process of degrading DDVP. TaPon1- like contained an open reading frame of 1317 bp, and the deduced amino acid sequence shared 21% homology with HuPON1, which also exhibits excellent hydrolysis of organophosphate-oxons compounds. By analysis of gene knockout, we found the Δ TaPon1- like knockout strain KO1 lost 35.6% of its DDVP-degradation capacity at 24 h, but this loss of degradation activity was recovered when the gene was complemented. Furthermore, the purified recombinant protein reTAPON1-LIKE, could transform DDVP only to dimethyl phosphate and showed significant paraoxonase activity (1028 U L ⁻¹ ). The reTAPON1-LIKE enzyme showed a broad degradation spectrum, degrading not only DDVP but also organophosphate-oxons and lactone. The kinetic parameters ( Km and kcat ) of the purified reTAPON1-LIKE were determined to be 0.23 mM and 204.3 s ⁻¹ for DDVP, respectively. The highest activity was obtained at 35 °C, and the optimal pH was 8.5. The activity of reTAPON1-LIKE was enhanced most significantly when 1.0 mM Ca ²⁺ was added but declined when 1.0 mM Cu ²⁺ was added. These results showed TAPON1-LIKE play an important role for DDVP degradation in the first step by T23 and provided clue to comprehensively understanding the degradation mechanism of organophosphate-oxons pesticides by filamentous fungi. Importance: The large amounts of residues of organophosphate pesticides in agroecological system has become a great threat to the safety of environment and humans. Bioremediation in association with microbial is innovative technology having a potential to alleviate such pollution problems. The genus Trichoderma is genetically diverse with capabilities to degrade chemical pesticides among different strains with agricultural significance. As a typical organophosphorus pesticide, it is one of the most employed compounds of the family. Though it was classified as a highly toxic pesticide by WHO due to its hazardous properties, it plays an important role in the control of plant pests, food storage and homes, as well as to treat infections in livestock. Therefore, we use DDVP as a model of organophosphate pesticide to study the mechanism of Trichoderma degrading organophosphate pesticides, for the aim of globally understanding molecular mechanism of enzymatic degradation of organophosphate pesticides by beneficial fungi.

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.