Abstract
The Dicer protein is one of the most important components of RNAi machinery because it regulates the production of small RNAs (sRNAs) in eukaryotes. Here, Dicer1-like gene (Pit-DCL1) and Dicer2-like gene (Pit-DCL2) RNAi transformants were generated via pSilent-1 in Penicillium italicum (Pit), which is the causal agent of citrus blue mold. Neither transformant showed a change in mycelial growth or sporulation ability, but the pathogenicity of the Pit-DCL2 RNAi transformant to citrus fruits was severely impaired, compared to that of the Pit-DCL1 RNAi transformant and the wild type. We further developed a citrus wound-mediated RNAi approach with a double-stranded fragment of Pit-DCL2 generated in vitro, which achieved an efficiency in reducing Pi-Dcl2 expression and virulence that was similar to that of protoplast-mediated RNAi in P. italicum, suggesting that this approach is promising in the exogenous application of dsRNA to control pathogens on the surface of citrus fruits. In addition, sRNA sequencing revealed a total of 69.88 million potential sRNAs and 12 novel microRNA-like small RNAs (milRNAs), four of which have been predicated on target innate immunity or biotic stress-related genes in Valencia orange. These data suggest that both the Pit-DCL1 and Pit-DCL2 RNAi transformants severely disrupted the biogenesis of the potential milRNAs, which was further confirmed for some milRNAs by qRT-PCR or Northern blot analysis. These data suggest the sRNAs in P. italicum that may be involved in a molecular virulence mechanism termed cross-kingdom RNAi (ck-RNAi) by trafficking sRNA from P. italicum to citrus fruits.
Highlights
Citrus fruit production exceeded 140 million tons in 2016, accounting for one-fifth of the total fruit production worldwide [1]
Dicer-like proteins regulate growth and conidiation in Colletotrichum gloeosporioides, and the double mutation abrogated the ability to penetrate Hevea brasiliensis [13]. These findings suggest that filamentous fungi employ RNAi-related pathways in various physiological processes to adapt to different environmental conditions; the RNAi machinery has never been reported in P. italicum, it is the most serious pathogen causing the decay of citrus fruits at the postharvest stage
Our study revealed the potential linkage between the RNAi machinery and the pathogenicity of P. italicum, and highlighted the probability of utilizing RNAi as a tool to control
Summary
Citrus fruit production exceeded 140 million tons in 2016, accounting for one-fifth of the total fruit production worldwide [1]. Citrus fruits are commercially handled in packing houses in order to maintain their postharvest quality, to increase their shelf life, as well as to reduce losses due to pathogen infection. Citrus blue mold, caused by Penicillium italicum, is one of the most destructive pathogens of postharvest citrus fruits, resulting in losses of up to 80% [2]. Advancing our knowledge regarding the molecular mechanisms underlying interactions between citrus fruit and P. italicum is profitable to reduce postharvest losses. Regulating the ambient pH of the host is considered to be one of the pathogenic mechanisms of Penicillium species. These pathogens can produce organic acids and deplete ammonium to acidify the ambient environment of citrus fruits
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