Identification And Characterization Of A Novel Circular RNA In Botryosphaeria Dothidea

The fungus Botryosphaeria dothidea(B.dothidea)is a causal agent of stem cankers,stem warts,and fruit ring rot,of pears worldwide,which reduces the quantity and quality of fruits and results in substantial economic losses.In our previous studies,two isolates of B.dothidea based on virulence assays on pear branches(var.huangguan)were denoted as virulent strain MAO-2 and hypovirulent strain XA-3.Transcriptome analysis revealed that XA-3 is a natural host of circular RNAs.However,the biological impact of circRNAs on the host is unknown.This study aims to insight the relationship between B.dothidea and circRNA along with its role in virulence.The key findings are as follows:A single-stranded(ss)circRNA(307 nucleotides)was identified preliminary from XA-3 through RT-PCR,and it was given the name B.dothidea circRNA 12944(BdCR12944).The secondary structure of BdCR12944 adopts a complex branched conformation and highly paired as predicted through the CLC Main Workbench v.21.0.4and comprises 72 adenine,55 uracil,78 guanine,and 102 cytosine nucleobases,with 58.6%guanine and cytosine(%GC)content.BLASTn analysis reveals that BdCR12944 showed no detected identities to any other reported RNA sequences from any other organisms,suggesting that it is a novel exogenous(ss)circRNA.The monomeric cDNAs were cloned into pGEM-T easy vector in two different orientations and subjected to in vitro transcription after linearization with a restriction enzyme to produce the probes labeled with digoxigenin.With the digoxigenin-labeled probes,the plus and minus polarity of BdCR12944 was determined with northern blot since it accumulates in a higher concentration than the counterpart in XA-3,and the circular nature of BdCR12944 was further confirmed by two-dimensional electrophoresis combined with northern-blot,indicating that a single nucleic acid band migrated slower than the fungal RNAs on PAGE gel.The dimeric cDNAs of BdCR12944 were in vitro transcribed and transfected the protoplasts of MAO-2 through Polyethylene glycol(PEG)mediated transformation.Twenty-four protoplast-generated colonies were identified by RT-PCR and dot blot hybridization,and results revealed that 11 sub-isolates were found positively transfected.While qualitatively through RT-qPCR,it was identified that transfectant-12 has the highest titer(6.88E+04 copies/?L)of BdCR12944.The inheritance ability of BdCR12944 from parents to their progenies was analyzed with a dot blot hybridization and absolute RT-qPCR to access the titer changes between the 1^st-subculture and the 10^th in vivo.The results showed that BdCR12944 had a fluctuant titer while no apparent difference between both generations.It suggests that BdCR12944 could be stably inherited from parent fungal cultures to their progenies.The biological effect of BdCR12944 on the host fungus was analyzed with the transfected sub-isolates of MAO-2 along with the control strains(XA-3 and MAO-2)on potato dextrose agar(PDA)media or amended with different cellular stress agents,including those involved in cell wall disruption(0.04%SDS),oxidative stress(0.05%H₂O₂),osmotic stress(1.5 M/L Na Cl,1 M/L KCl,0.5 M/L CaCl₂,and 1 M/L glucose).On PDA media,the growth rate and biomass production of transfectants were significantly reduced as compared with these of MAO-2(9.38±0.209 to 11.44±0.096 via 12.52±0.127mm/day,0.164±0.004 to 0.262±0.007 g/day via 0.286±0.010 g/day,respectively);while their morphologies were not apparently changed except for transfectant-12 similar to that of XA-3.On PDA amended with varied cellular stress agents,their growth rates were enhanced by glucose and H₂O₂,reduced by Na Cl,while no obviously affected by the remaining chemicals(KCl,CaCl_2,and SDS).As accessed their virulence on pear fruits and branches(var.huangguan)at 4 and 10 dpi,respectively.The results showed that transfectants induced no lesions on the branches,while MAO-2 induced the lesions with lengths of 28.42±14.67 mm;the transfectants caused the lesions on fruits,with the sizes(24.85±11.14 mm to 37.71±16.78 mm)almost similar to the XA-3 but significantly less than these by the MAO-2 did.The results depicted that the transfectants lost and attenuated their virulence on pear branches and fruits,respectively,after transfected by BdCR12944,suggesting that BdCR12944 induced hypovirulence in transfected isolates of MAO-2.To check whether BdCR12944 can infect other fungi,the protoplasts of Diaporthe eres(DK-362),Alternaria alternata(GS-18),and Colletotrichum fructicola(ZGTL)were produced and transfected with dimeric RNAs transcribed in vitro based on the dimeric cDNAs of BdCR12944 and subjected to detection by dot blot hybridization and RT-PCR analysis.It reveals that novel BdCR12944 could infect D.eres(DK-362)and A.alternata(GS-18)with infection rates of 29.1%and 20.83%,respectively,while not C.fructicola(ZGTL).Moreover,to check whether BdCR12944 can infect or transmit from the mycelia to plants,Nicotiana benthamiana plants were inoculated with their dimeric RNAs and positive transfectant-12,respectively,and indexed by northern blot.The results showed that no matter from the inoculated RNAs or the mycelia,BdCR12944 could spread into newly developed leaves and replicate there,and vice versa,i.e.,BdCR12944 could be delivered from N.benthamiana to MAO-2 as inoculated on the seedlings.To check whether BdCR12944 modulates the host metabolism process,transfectant-12 was subjected to transcriptome analysis together with MAO-2,and 754 differential expressed genes(DEGs),with 433 and 311 up-and down-regulated,respectively,were identified.According to GO annotation,25 genes involved in the cellular components,98biological processes,and 92 molecular functions out of 311 down-regulated DEGs.The down-regulated genes were significantly enriched in the pathway of peptidase activity,and their expression tendency(with ten down-regulated DEGs)was further verified relative qRT-PCR assays.Collectively,a novel circular RNA,named BdCR12944,was identified in the mycelia of B.dothidea and showed it could independently replicate and transfects other fungi and even plants.BdCR12944 plays regulatory roles in the biological traits of the host fungi,including the morphologies,growth rates,and virulence,and it may contribute to understanding circular RNA in fungi by providing substantial knowledge.