Jin Wu*,Shengling YinLi LinDongxio LiuSicho RenWenjing ZhngWencheng MengPeipei ChenQinfu Sun,Yujie Fng,Cunxu WeiYouping Wng,*

a Key Laboratory of Plant Functional Genomics of the Ministry of Education,Yangzhou University,Yangzhou,China

b Jiangsu Key Laboratory of Crop Genomics and Molecular Breeding,Yangzhou University,Yangzhou,China

Keywords:Sclerotinia sclerotiorum Brassica napus SIGS HIGS RNAi

ABSTRACT Sclerotinia sclerotiorum is generally considered one of the most economically damaging pathogens in oilseed rape (Brassica napus).Breeding for Sclerotinia resistance is challenging,as no immune germplasm available in B.napus.It is desirable to develop new breeding strategies.In the present study,hostinduced gene silencing (HIGS),developed based on RNA interference (RNAi),was applied to protect B.napus from S.sclerotiorum infection.Three pathogenicity genes,the endo-polygalacturonase gene(SsPG1),cellobiohydrolase gene (SsCBH),and oxaloacetate acetylhydrolase gene (SsOAH1),were chosen as HIGS targets.Co-incubation of synthesized double-stranded RNAs(dsRNAs)with S.sclerotiorum in liquid medium significantly reduced the transcript levels of the target genes.Application to plant surfaces of dsRNA targeting the three genes conferred effective protection against S.sclerotiorum.Stable transgenic B.napus plants expressing small interfering RNAs with sequence identity to SsPG1,SsCBH,and SsOAH1 were generated.HIGS transgenic B.napus prevented the expression of S.sclerotiorum target genes,slowed pathogenicity-factor accumulation,impeded fungal growth,and suppressed appressorium formation,thereby conferring resistance to S.sclerotiorum.Simultaneous silencing of SsPG1,SsCBH,and SsOAH1 by stable expression of a chimeric hairpin RNAi construct in B.napus led to enhanced protection phenotypes(with disease lesion size reduced by 36.8%–43.7%).We conclude that HIGS of pathogenic-factor genes of S.sclerotiorum is a promising strategy for controlling Sclerotinia rot in oilseed rape.

1.Introduction

Sclerotinia rot,caused by the necrotrophic fungus Sclerotinia sclerotiorum,is one of the most devastating diseases of oilseed rape(Brassica napus)worldwide.In Canada,yearly average incidences of Sclerotinia rot often range from 10% to 20%,representing an approximate average yield loss of 5%–10%[1].In China,Sclerotinia rot causes annual yield losses of 10%–20%,and losses as high as 80% occur in severely infected fields [2].Fungicides are widely applied to manage this fungal disease,but determining the optimal application time is difficult,thus often resulting in ineffective sprays and environmental contamination.The most economical,effective,and eco-friendly way to control Sclerotinia rot is by breeding and cultivating resistant oilseed rape cultivars.However,no immune germplasm of B.napus or its closely related species has been identified to date.Quantitative trait locus(QTL)mapping and genome-wide association studies have shown that Sclerotinia rot resistance involves complex genetic underpinnings determined by multiple minor QTL [3–6].No QTL have been cloned,limiting their utilization in Sclerotinia rot resistance breeding.It is desirable to develop new approaches to protect oilseed rape from S.sclerotiorum.

RNA interference(RNAi),a powerful genetic tool used to assess gene function by interfering with endogenous gene expression at the transcriptional or posttranscriptional level,is considered a promising approach for plant protection via host-induced gene silencing(HIGS)[7]or host-delivered RNAi[8].HIGS silences genes in pathogens or pests by in planta expression of double-stranded RNAs (dsRNAs) or hairpin RNAs complementary to essential and/or pathogenicity-associated genes of pathogens or pests,thereby conferring engineered plant protection against infection or predation.This strategy was initially applied to promote crop resistance to root-knot nematodes[9]and insects[10,11].Subsequently,HIGS also proved to be a promising strategy for preventing disease incited by filamentous fungi,including fungi[7,12]and oomycetes[13].Proof of concept for HIGS in filamentous fungi was demonstrated by Tinoco et al.[12]and Nowara et al.[7].An in vivo interference phenomenon in the phytopathogenic fungus Fusarium verticillioides was reported [12],in which the expression of the βglucuronidase (GUS) gene was specifically silenced by inoculation with mycelial cells of transgenic tobacco plants engineered with a GUS gene-inferring cassette [hairpin (hp) GUS].The expression of dsRNAs or antisense RNAs targeting fungal transcripts in barley(Hordeum vulgare) and wheat (Triticum aestivum) inhibited the development of powdery mildew fungi [7].In the last decade,numerous studies have demonstrated the effectiveness of HIGS for controlling other fungal diseases,including wheat stripe rust[14,15],leaf rust [16,17] and head blight [18–21];barley head blight [22,23];cotton Verticillium wilt [24,25];soybean rust[26];tomato gray mold[27]and Verticillium wilt[28];and lettuce downy mildew [13].

Somewhat unexpectedly,recent studies have shown that direct spraying of dsRNAs or small RNAs (sRNAs) that target essential pathogen genes onto plant surfaces confers effective crop protection,a phenomenon that has been termed spray-induced gene silencing (SIGS) [29].The first successful application of SIGS was reported by Koch et al.[30],who showed that spraying barley leaves with a 791-nt dsRNA targeting three Fusarium graminearum ergosterol-biosynthesis genes(CYP51A,CYP51B,and CYP51C)effectively inhibited fungal infection.Wang et al.[27] reported that spraying sRNAs or dsRNAs targeting the Dicer-like protein 1(DCL1)and DCL2 genes of Botrytis cinerea onto the surfaces of some fruits,vegetables,and flowers inhibited gray mold disease.Cumulative evidence[27,30,31]suggests that dsRNAs and sRNAs sprayed on plant surfaces can either be taken up by fungal cells directly or accumulate in plant cells before being transferred into fungal cells.

Thus,HIGS and SIGS offer unprecedented potential for crop protection against fungal pathogens [32].However,these strategies are not effective for preventing all fungal diseases,as exemplarily shown by the insensitivity of Zymoseptoria tritici to both HIGS and SIGS [33].Andrade et al.[34] showed that HIGS of the chitin synthase gene of S.sclerotiorum generated white mold-tolerant tobacco plants.Exogenous application of dsRNA protected B.napus plants from S.sclerotiorum [35].These studies demonstrated that the HIGS and SIGS strategies are effective against S.sclerotiorum and greatly boost our confidence in the utilization of HIGS to protect B.napus from S.sclerotiorum.

Target gene selection is crucial for HIGS,and genes essential for fungal growth or pathogenicity are the most common choices.For S.sclerotiorum,the molecular aspects of pathogenicity are concentrated mainly on the contributions of cell wall-degrading enzymes(CWDEs) and oxalic acid (OA),which are secreted to facilitate colonization by pathogen cells and degradation of the host cell wall[36–38].The objective of the present study was to determine the effect of silencing of three pathogenic genes of S.sclerotiorum by SIGS and HIGS on resistance to the pathogen.

2.Materials and methods

2.1.Plant and fungal materials and growth conditions

The B.napus line J9712,kindly provided by Prof.Yongming Zhou of the National Key Laboratory of Crop Genetic Improvement,Huazhong Agricultural University,Wuhan,Hubei,China,was used as a transformation recipient.For cotyledon inoculation,both J9712 and the transformed lines were grown in growth chambers under a 16 h light/8 h dark photoperiod at 24 °C,and a relative humidity of 60%.For leaf and stem inoculation,all plants were grown in the experimental field at Yangzhou University,Jiangsu,China.The single-target HIGS lines were grown in three consecutive growing seasons,2018–2019 (T1),2019–2020 (T2) and 2020–2021 (T3).The T1generation of triple-target HIGS lines was grown in the 2020–2021 growing season.All field experiments were laid out in a randomized complete block design with three replications.The S.sclerotiorum isolate SS-1 was maintained and cultured on potato dextrose agar(PDA,Becton,Dickinson and Company,Franklin Lakes,NJ,USA),as described previously [39].

2.2.Construction of an HIGS vector and plant transformation

A DNA sequence containing intron 2 of the pyruvate orthophosphate dikinase (PDK) gene from Flaveria trinervia (GenBank:X79095.1767 bp)and multiple restriction endonuclease sites were synthesized by Sangon Biotech (Shanghai) Co.,Ltd.(Shanghai,China)and cloned into the binary plant expression vector pMDC83[40]digested with XbaI and SacI to establish the intron-containing hairpin vector pMDC83-ihpRNAi.

Actively growing hyphae of the S.sclerotiorum isolate SS-1 on PDA were harvested and stored at-80°C.Total RNA was extracted using a Fungal Total RNA Isolation Kit(Sangon Biotech)and treated with DNase I.First-strand cDNA was synthesized with the Transcriptor First Strand cDNA Synthesis Kit (Roche,Mannheim,Germany) according to the manufacturer’s instructions,and cDNA was used as a template for gene cloning.The complete open reading frames of SsPG1,SsCBH,and SsOAH1 were cloned from the SS-1 isolate using gene-specific primers (Table S1) designed from the reference genome sequence of S.sclerotiorum strain 1980 [41].

For single-target HIGS constructs,the 381 bp fragment of SsPG1,351 bp fragment of SsOAH1,and 425 bp fragment of SsCBH were PCR-amplified using predesigned primers (Table S1).For the triple-target HIGS construct,a 750 bp tandem DNA fragment containing three 250-bp DNA fragments from SsPG1,SsOAH1 and SsCBH was synthesized by Sangon Biotech.Each fragment was cloned into the pMDC83-ihpRNAi vector in the sense and antisense orientations (sense-intron-antisense cassette) via homologous recombination (ClonExpress II One Step Cloning Kit,Vazyme,Nanjing,Jiangsu,China).

The HIGS constructs were transformed into Agrobacterium tumefaciens strain GV3101 by electroporation and then transformed into J9712 using the A.tumefaciens-mediated hypocotyl method as described by Dai et al.[42].Positive transgenic B.napus plants were identified by PCR with specific primers (Table S1).

2.3.Synthesis and application of dsRNAs in liquid medium and on plant surfaces

Approximately 350–450 bp dsRNAs complementary to each of the selected genes were synthesized using a T7 RNAi Transcription Kit (Vazyme) according to the manufacturer’s instructions.The T7 promoter sequence was introduced at both the 5′and the 3′ends of the RNAi target fragments via PCR.The DNA fragments containing the T7 promoter were purified and used for the amplification of dsRNA.As a control,a 322 bp green fluorescent protein (GFP)-dsRNA was also synthesized using the pMDC83 plasmid as a template.The synthesized dsRNAs were quantified using a NanoDrop spectrophotometer (NanoDrop,Wilmington,DE,USA) and stored at -80 °C.

A plug 1 mm in diameter was cut from the actively growing margin of a 2-day-old colony,transferred to a dish (6 cm in diameter)containing 3 mL of potato dextrose broth(PDB,Becton,Dickinson and Company),and cultured at 23 °C for 48 h on a platform shaker at 25 r min-1.Then,dsRNAs were applied at a dose of 500 ng mL-1according to a previous study[35]and cultured under the same conditions.Tissues were harvested at 0,24,48,72,and 96 h post-treatment(hpt)to detect the expression patterns of target genes.Three independent biological replicates were tested.

To determine whether the direct application of dsRNAs to plant surfaces could confer efficient protection against S.sclerotiorum,an infection assay was performed on detached Nicotiana benthamiana leaves.The latest or penultimate fully extended leaves of similar size were excised from 30-day-old N.benthamiana plants grown in growth chambers (16 h light/8 h dark photoperiod,24 °C).Detached N.benthamiana leaves (approximately 50 leaves) were treated with 25 μL of dsRNA (10 ng μL-1) and 0.015% Silwet L-77.After the dsRNA solution dried for about 20 min,mycelial agar plugs 5 mm in diameter punched from the growing margin of a 2-day-old S.sclerotiorum culture were inoculated at the same position used for dsRNA treatment.The leaves were placed in a plastic tray(61×41×9 cm)containing wet gauze on the bottom,and the tray was covered with a plastic film to maintain high humidity.The plastic trays with inoculated leaves were stored at 23 °C in the dark.Fungal lesion size was measured at 24 h post-inoculation(hpi) using ImageJ software (https://imagej.nih.gov/ij/).The GFPdsRNA was used as a control.

2.4.Assessment of resistance to S.sclerotiorum

Resistance of HIGS transgenic plants to S.sclerotiorum was assessed by three inoculation procedures:detached-leaf,stem,and cotyledon inoculation.For each transgenic line,approximately 20 plants in each of the three replicates were assessed by detached-leaf and stem inoculation as described previously [39],following Zhao and Meng [43].The nontransgenic line J9712 and the transgenic negative line were used as controls.To determine the expression of S.sclerotiorum target genes for silencing,tissues extending 10 mm beyond the inoculation site on the leaves of HIGS transgenic and J9712 plants were harvested at 24 hpi and stored at-80 °C.

Cotyledon inoculation was performed as described by Garg et al.[44] with minor modifications.Seeds were germinated on wet filter paper with 100 mg L-1hygromycin.Normally germinating seedlings (positive transgenic plants) were transferred to soil and grown in growth chambers until the cotyledons were fully expanded.Six mycelial agar plugs 5 mm in diameter were cut from the actively growing margin of a 2-day-old colony,transferred to a 250-mL flask containing 200 mL of PDB,and cultured at 23°C on a platform shaker at 150 r min-1.After two days,mycelial balls were harvested and washed twice with sterile distilled water.The obtained mycelial balls were transferred into 100 mL of PDB and then smashed by high-speed homogenization (ULTRA-TURRAX T18 digital,IKA,Staufen,Germany) at 10,000 r min-1for 15 min.The mycelial suspension was then filtered through degreased gauze and adjusted to a concentration of 105fragments mL-1with PDB using a hemocytometer.Mycelial suspension (10 mL) was deposited onto each cotyledon lobe using a micropipette.The inoculated cotyledons were kept under the same conditions as described for the N.benthamiana leaf inoculation.Fungal lesion size was measured at 96 hpi with ImageJ.This assay was conducted with three replications.

2.5.Small RNA sequencing

Small-RNA sequencing was performed to confirm the presence of target gene-specific sRNAs in HIGS transgenic plants.For each HIGS construct,uninoculated leaves of five transgenic T1lines(three plants for each line) were randomly selected and mixed for small RNA sequencing.Total RNA was extracted with TRIzol reagent (Invitrogen,Carlsbad,CA,USA).One microgram of total RNA was used for library construction with a TruSeq Small RNA Library Preparation Kit (Illumina,San Diego,CA,USA) according to the manufacturer’s instructions.All samples were sequenced using an NovaSeq 6000 sequencing system (Illumina),producing 50 single-end reads.The original data set was deposited into the NCBI Sequence Read Archive (SRP308286).The low-quality and adapter sequences were removed with cutadapt [45].Clean reads were then mapped to the coding sequences of target genes with Bowtie v.1.2.3 [46] using the default parameters.sRNA mapping figures were generated using igvtools (http://software.broadinstitute.org/software/igv/igvtools).

2.6.qRT-PCR analysis of the target genes in S.sclerotiorum

The transcript expression of each target gene was assessed by qRT-PCR.Total RNA extraction and cDNA synthesis were performed as described above.qRT-PCR was performed on a StepOne-Plus fluorescence quantitative system (Applied Biosystems,Foster City,CA,USA) using SYBR Green Real-Time PCR Master Mix(Thermo Fisher,Waltham,MA,USA).The S.sclerotiorum betatubulin gene Sstub1(SS1G_04652)was used as an endogenous control.For each target gene,qRT-PCR assays were repeated at least three times,each with three biological replicates.The primers used for the qRT-PCR assays are listed in Table S1.

2.7.OA measurement

To determine whether the in vitro silencing of SsOAH1,a key gene responsible for OA biogenesis and accumulation [47,48],could inhibit OA biogenesis,the OA accumulation in liquid medium and inoculated leaves after SsOAH1-dsRNA treatment was quantified.Culture filtrates of S.sclerotiorum were collected at 0,12,24,36,48,60,and 72 h after SsOAH1-dsRNA treatment and centrifuged at 8000 r min-1for 5 min,and 25 μL of the supernatant was used for the assay.To quantify in planta OA accumulation,tissues extending 10 mm beyond the inoculation site on the leaves were harvested at 12 and 24 hpi and then ground into powder with liquid nitrogen.The powder (0.5 g) was rapidly homogenized in 350 μL of cold oxalate assay buffer.The homogenate was incubated for 10 min on ice and centrifuged at 8000 r min-1for 5 min to remove insoluble material,and 25 μL of the supernatant was then used for the assay.The concentration of OA was determined with an Oxalic Acid Colorimetric Assay Kit (Sigma-Aldrich,St.Louis,MO,USA).Each OA measurement experiment was conducted with three replications.

2.8.Cotton blue staining

S.sclerotiorum-challenged cotyledons were excised at 4 and 20 hpi.The sampled cotyledons were decolorized in an acetic acid:ethanol (2:1,v/v) solution at room temperature for 48 h and then washed twice with sterile distilled water.The cotyledons were stained with a cotton blue solution (phenol 20 g,lactic acid 20 mL,glycerin 40 mL,cotton blue 0.05 g,distilled water 20 mL)overnight.All samples were observed for blue staining under a light microscope (BX53,Olympus,Tokyo,Japan).

2.9.Statistical analysis

Statistical analysis was performed with SPSS 19.0 (IBM SPSS Statistics,New York,NY,USA).Graphs were drawn with Microsoft Excel 2019 (Microsoft,Redmond,Washington,USA).

3.Results

3.1.Selection of HIGS target genes

In a previous study[49],we performed dynamic transcriptomic analyses to identify differential defense responses to S.sclerotiorum in a resistant line(J964)and a susceptible line(J902)of B.napus at 24,48 and 96 hpi.In all inoculated samples,there were 28.2 million reads of S.sclerotiorum origin,equal to a 161× sequencing depth of the S.sclerotiorum transcriptome (15.8 Mb).In total,10,040 and 9,626(66%–69%of the annotated genes in the S.sclerotiorum genome)were expressed during the S.sclerotiorum infection of J902 and J964,respectively(fragments per kilobase of transcript per million fragments mapped(FPKM)≥1 at any point).Two genes encoding CWDEs (an endo-polygalacturonase gene,SsPG1,and a cellobiohydrolase gene,SsCBH) and an oxaloacetate acetylhydrolase gene (SsOAH1) associated with OA accumulation were identified as having the highest expression among all the expressed genes (mean FPKMs of all the inoculated samples were 20768,8271,and 6623,respectively,and the mean FPKM of all genes was 180.4) (Fig.1A–C).All three genes were dramatically upregulated over the three inoculation time points in both lines(Fig.1A–C).As CWDEs and OA are pathogenicity factors of S.sclerotiorum,the two CWDEs encoding genes (SsPG1 and SsCBH) and the OA biosynthesis gene (SsOAH1) are presumably essential for the pathogenicity of S.sclerotiorum and have potential as targets for controlling S.sclerotiorum by HIGS.Based on the reference genome sequence,the complete open reading frames of SsPG1(SS1G_10167),SsOAH1 (SS1G_08218),and SsCBH (SS1G_09020)were cloned from S.sclerotiorum isolate SS-1 (Fig.S1).

3.2.In vitro silencing of S.sclerotiorum target genes

Synthesized dsRNAs complementary to each of the selected genes were applied to S.sclerotiorum growing in axenic liquid medium.The transcript levels of SsPG1 and SsCBH were significantly reduced by 56% and 65%,respectively,at 24 hpt compared to 0 hpt,and the level of suppression persisted for 72 hpt (Fig.1D,F),while the expression of SsOAH1 was only slightly downregulated at 24 hpt and was inhibited significantly from 48 to 96 hpt (reduced by approximately 70%,Fig.1E).Thus,all three synthesized dsRNAs delivered in vitro silenced the expression of their target genes in S.sclerotiorum,and the dsRNA-mediated gene silencing lasted for up to 48 h.

Fig.1.Silencing S.sclerotiorum target genes by application of synthesized dsRNAs in liquid medium.(A–C) Expression patterns of S.sclerotiorum pathogenicity-associated genes encoding endo-polygalacturonase(SsPG1),oxaloacetate acetylhydrolase(SsOAH1)and cellobiohydrolase(SsCBH)in a resistant line(J964)and a susceptible line(J902)of B.napus at 24,48 and 96 h post-inoculation(hpt)as revealed by transcriptomic analysis.FPKM,fragments per kilobase of transcript per million fragments mapped.(D–F)Expression levels of target genes after application of 500 ng mL-1 dsRNA in vitro as determined by qRT-PCR at 0,24,48,72,and 96 hpt.For each qRT-PCR assay,the expression data were normalized to the endogenous control gene Sstub1,and relative expression values were then calculated based on the GFP-dsRNA control.(G–H) pH and OA accumulation in liquid medium used to co-culture S.sclerotiorum and SsOAH1-dsRNA or GFP-dsRNA (control).Error bars in (D–H) indicate standard deviations of three biological replicates.Significance of comparison with 0 hpt in (D–F) and with GFP-dsRNA (control) in G–H (*, P ＜0.05;**, P ＜0.01,Student’s t-test).

To determine whether the in vitro silencing of SsOAH1 by dsRNA can inhibit OA biogenesis in S.sclerotiorum,the pH was monitored and OA accumulation in liquid medium was quantified.As the S.sclerotiorum cultivation time extended (0–72 h),OA accumulated gradually and the pH value decreased correspondingly (Fig.1G,H).The OA content clearly reflected lower accumulation,and the pH of the medium decreased more slowly after the application of SsOAH1-dsRNA (Fig.1G,H),suggesting that externally applied dsRNA inhibited pathogen pathogenicity-factor biosynthesis.

3.3.SIGS reduces S.sclerotiorum infection in N.benthamiana

To determine whether direct application of synthesized dsRNAs on plant surfaces could confer protection against S.sclerotiorum,N.benthamiana leaves were treated with 25 μL (10 ng μL-1) of dsRNAs,and S.sclerotiorum was inoculated at the same position after the dsRNAs solution dried.By 24 hpi,the N.benthamiana leaves treated with dsRNAs targeting SsPG1,SsOAH1 and SsCBH showed much smaller lesions than the controls.Lesion sizes were reduced by 47.2%,58.3% and 84.6%,respectively,compared to those of control leaves treated with GFP-dsRNA (Fig.2A,B).Thus,application onto plant surfaces of dsRNAs targeting the pathogenicity-associated genes SsPG1,SsCBH,and SsOAH1 inhibited S.sclerotiorum infection.

3.4.Construction of HIGS vectors and confirmation of target genespecific sRNA expression

Encouraged by the in vitro results,we next investigated whether expressing dsRNAs in B.napus could protect transgenic plants from S.sclerotiorum infection.To this end,we developed an intron-containing hairpin vector (pMDC83-ihpRNAi) based on the binary plant expression vector pMDC83.The pMDC83-ihpRNAi vector contained a hygromycin phosphotransferase selection marker gene,a double CaMV 35S promoter,a spacer sequence(PDK intron),and a nopaline synthase terminator (Fig.3A).The HIGS target regions of SsPG1,SsOAH1,and SsCBH were the same as those in the in vitro tests (381 bp of SsPG1,351 bp of SsOAH1,and 425 bp of SsCBH),and these sequences were inserted into the pMDC83-ihpRNAi vector to generate the HIGS constructs.As predicted in silico using the SI-FI software tool (http://labtools.ipk-gatersleben.de/),no putative off-targets of the HIGS constructs were found in either the S.sclerotiorum or B.napus transcriptomes.The HIGS constructs SsPG1-RNAi,SsOAH1-RNAi,and SsCBH-RNAi were transformed into the pure B.napus line J9712,generating respectively 25,11,and 21 T0positive transformants.

To determine the presence of complementary small interfering RNAs(siRNAs)in transgenic SsPG1-RNAi,SsOAH1-RNAi,and SsCBHRNAi plants,small-RNA sequencing was performed on plants of the T1generation.For each RNAi construct,five transgenic T1lines were randomly selected and mixed for small RNA sequencing.The siRNAs derived from the SsPG1-RNAi,SsOAH1-RNAi and SsCBHRNAi constructs were present in the transgenic T1lines(Fig.3B–D).These S.sclerotiorum gene-specific siRNAs were distributed across the target gene regions (Fig.3B–D).The most abundant siRNAs from all three of the HIGS constructs were 21–22 nt in length,followed by 24,20,and 23 nt(Fig.3E).Most strikingly,the expression abundances of the specific siRNAs in SsPG1-RNAi,SsOAH1-RNAi and SsCBH-RNAi transgenic plants were very high,accounting for respectively 0.45%,0.23%,and 0.05% of the total small RNAs detected in each library(Fig.3E).Thus,these HIGS constructs were expressed in transgenic B.napus plants.

3.5.HIGS in B.napus confers resistance to S.sclerotiorum

Resistance was assessed by detached-leaf inoculation with S.sclerotiorum mycelial agar plugs in T2transgenic lines,including five independent SsPG1-RNAi lines,three independent SsOAH1-RNAi lines,and three independent SsCBH-RNAi lines (Fig.4A).All the transgenic lines exhibited significantly reduced areas of S.sclerotiorum disease lesions compared with those of the nontransgenic line J9712 (Fig.4A,B).The disease lesion areas at 48 hpi were reduced by 26.7%–38.7% for the SsPG1-RNAi lines,24.7%–34.4%for the SsOAH1-RNAi lines and 20.8%–26.8% for the SsCBH-RNAi lines (Fig.4B).Similar results were obtained for the T3generation,with both the nontransgenic and transgenic negative lines serving as controls (Fig.4C),indicating that the resistance trait was successfully transmitted to subsequent generations.

To further confirm that reduced S.sclerotiorum infection was caused by the silencing of target genes,we evaluated the transcript levels of these genes in S.sclerotiorum-inoculated leaves of HIGS transgenic plants (T2) and nontransgenic plants at 24 hpi.The relative expression levels of SsPG1,SsOAH1 and SsCBH in the transgenic lines PG1-RNAi-7,OAH1-RNAi-7,and CBH-RNAi-9 were reduced to 48.9%,33.9%,and 7.6%,respectively,of those in nontransgenic leaves(Fig.4D).Furthermore,the decreased expression of SsOAH1 in S.sclerotiorum on the leaves of SsOAH1-RNAi transgenic plants during infection resulted in lower OA accumulation than in nontransgenic leaves at both 12 and 24 hpi (Fig.4E).

Besides the clear resistance reaction in the leaves of HIGS transgenic seedlings,cotyledon and stem resistance were also observed in respectively seedling and mature plants of the HIGS transgenic lines (Fig.5).The lesion area on cotyledons at 96 hpi was reduced by 41.1%–56.9% in the SsPG1-RNAi lines,47.7%–61.8% in the SsOAH1-RNAi lines,and 37.1%–50.1%in the SsCBH-RNAi lines compared to the nontransgenic cotyledons(Fig.5C,D).A stronger resistance reaction was observed in cotyledons than in stems because cotyledons were inoculated with mycelial suspension,which contained fewer mycelia than leaf and stem inoculated with mycelial agar plugs,causing the HIGS transgenic plants to inhibit the S.sclerotiorum target genes more thoroughly.

Fig.2.SIGS-mediated control of S.sclerotiorum infection on N.benthamiana leaves treated with dsRNA.(A)Detached leaves of 30-day-old N.benthamiana plants were treated with 25 μL of dsRNAs(10 ng μL-1)targeting SsPG1,SsOAH1,SsCBH or GFP(control).After the dsRNA solution dried,S.sclerotiorum was inoculated onto the same position as the dsRNA,and fungal lesion size were evaluated at 24 hpi.Scale bars,1 cm.(B)Lesion sizes were measured.Error bars indicate standard errors of lesion sizes on 50 leaves.Asterisks represent statistically significant differences (*, P ＜0.05;**, P ＜0.01,Student’s t-test).

Fig.3.Structure of the HIGS construct and confirmation of target gene-specific siRNA expression by small-RNA sequencing.(A)Schematic of the HIGS construct.Sense(S)and antisense (AS) fragments were inserted between the double CaMV 35S promoter (2× 35S) and the nopaline synthase terminator (nos T),forming a sense-pyruvate orthophosphate dikinase (PDK) intron (IntPDK)-antisense cassette. Hygr,hygromycin phosphotransferase selection marker gene.(B–D) Small-RNA profiling of S.sclerotiorum target genes in uninoculated leaves of HIGS transgenic plants (T1).The target regions (296–676 bp of SsPG1,291–641 bp of SsOAH1,470–894 bp of SsCBH) are labeled with different colors.The coverage depth and alignment track were obtained by mapping the Illumina sequence reads to the coding sequences of target genes.(E) Length distribution and abundance of target gene-specific siRNAs.

Next,microscopic analyses were performed to identify differences in infection processes between HIGS transgenic lines and the nontransgenic line J9712.At 6 hpi,many hyphae were observed on the cotyledon surfaces of J9712 plants,whereas only a few hyphae appeared on the cotyledon surfaces of PG1-RNAi-7-1,OAH1-RNAi-7-3 and CBH-RNAi-3-2 plants (Fig.S2A–D).Fungal hyphae continued to grow on the cotyledon surfaces,and no observable differences in the number of hyphae were detected between the HIGS transgenic lines and J9712 (Fig.S2E–H).However,many complex appressoria formed on J9712 (Fig.S2I) but not on the HIGS transgenic lines at 20 hpi (Fig.S2J–L).Only a few finger-like simple appressoria were observed in the HIGS transgenic lines at 20 hpi(Fig.S2J–L).These results suggested that HIGS transgenic plants could suppress fungal growth and appressorium formation.

No significant differences in agronomic traits between the transgenic and J9712 plants were detected(Table S2).This finding suggested a low probability of off-target B.napus gene silencing,consistent with the SI-FI software prediction of no putative offtarget sequences of the HIGS constructs in B.napus.

3.6.Simultaneous silencing of three target genes by HIGS conferred increased resistance

To determine whether simultaneous HIGS of SsPG1,SsOAH1,and SsCBH could increase the efficiency of pathogen inhibition,we designed a chimeric hairpin RNAi construct targeting all three genes.A tandem DNA fragment containing three 250-bp DNA fragments,which were partial target regions of the SsPG1,SsOAH1 and SsCBH constructs designed for the single-target HIGS experiment,was inserted into the pMDC83-ihpRNAi vector (Fig.6A).

Fig.4.Assessment of disease resistance in HIGS-transgenic B.napus to S.sclerotiorum by the detached-leaf inoculation method.(A)Symptoms of S.sclerotiorum infection on B.napus leaves at 48 h post-inoculation(hpi).Scale bars,1 cm.(B–C)Quantification of the lesion areas of HIGS transgenic T2(B)and T3(C)B.napus lines,the nontransgenic line J9712,and the transgenic negative line at 48 hpi.Error bars represent standard error of the lesion sizes from 60 leaves of each line(20 leaves of each of the three replicates).(D)Expression levels of target genes in S.sclerotiorum-infected B.napus plants at 24 hpi.Values were normalized to the fungal endogenous control gene Sstub1(SS1G_04652).(E)Quantification of OA concentration in S.sclerotiorum-infected leaves of SsOAH1-RNAi transgenic and J9712 plants at 12 and 24 hpi.Error bars in D and E indicate standard deviations of three replicates.Significance compared with J9712 (*, P ＜0.05;**, P ＜0.01,Student’s t-test).

The chimeric RNAi construct was transformed into J9712 plants,generating 18 T0-positive transformants.Five triple-target HIGS transgenic T1lines were randomly selected and mixed for small-RNA sequencing.Specific siRNAs specific to the three S.sclerotiorum target gene regions were present in the transgenic T1lines(Fig.6B),showing that this chimeric RNAi construct functioned as expected.The expression abundances of the specific siRNAs of SsPG1,SsOAH1,and SsCBH were very high,accounting for 0.3% of the total small RNAs detected in the library.Quantitative RT-PCR analysis showed that the specific siRNAs generated from transgenic plants successfully silenced the three target genes of S.sclerotiorum in the inoculated leaves (Fig.6C).

The resistance of triple-target HIGS lines (T1) was assessed by both the detached-leaf inoculation and cotyledon inoculation methods.The leaves of triple-target HIGS lines exhibited significantly smaller (by 36.8%–43.7%) disease lesions than those of J9712 plants at 48 hpi (Fig.6D,E).The resistance of the tripletarget HIGS lines was also demonstrated in a cotyledon inoculation assay.The cotyledon lesion areas of the triple-target HIGS lines were reduced by 57.3%–60.4% compared to that on J9712 cotyledons at 96 hpi (Fig.6F,G).The resistance of the triple-target HIGS lines appeared to be stronger than that of the single-target RNAi lines.Thus,simultaneously silencing multiple target genes by HIGS conferred increased resistance to S.sclerotiorum in B.napus.

4.Discussion

The necrotrophic fungus S.sclerotiorum incurs large yield losses in oilseed rape as well as other oil crops,such as soybean (Glycine max) and sunflower (Helianthus annuus),worldwide.However,breeding Sclerotinia-resistant crops is challenging,owing to the limited availability of immune or highly resistant germplasm.In this study,we showed that HIGS using the transgenic expression of S.sclerotiorum gene-targeting dsRNAs was an effective strategy for controlling Sclerotinia rot in oilseed rape.

Fig.5.Assessment of the disease resistance of HIGS transgenic B.napus(T2)to S.sclerotiorum by the stem-inoculation method in the field and by the cotyledon-inoculation method in the growth chamber.(A,C)Symptoms of S.sclerotiorum infection on B.napus stems at 120 h post-inoculation(hpi)and cotyledons at 96 hpi.Scale bars,3 cm in(A)and 1 cm in(C).(B,D)Quantification of the lengths of lesions on stems at 120 hpi(B)and the lesion sizes of cotyledons at 96 hpi(D)on HIGS transgenic B.napus lines and the nontransgenic line J9712.Error bars represent standard error of the lesion size on 60 stems and cotyledons of each line (20 stems and cotyledons for each of the three replicates).Significance compared with J9712 (*, P ＜0.05;**, P ＜0.01,Student’s t-test).

Target gene selection is the most important prerequisite for HIGS.In general,genes that play a vital role in pathogen growth,development,and pathogenicity are considered candidate HIGS targets.The pathogenicity mechanism of S.sclerotiorum is complex and involves several factors including CWDEs,OA,and secretory effector proteins [50].In this study,two genes encoding CWDEs(SsPG1,and SsCBH) and an oxaloacetate acetylhydrolase gene(SsOAH1,associated with OA accumulation) showed the highest expression among all S.sclerotiorum genes expressed during infection (Fig.1A–C).The high expression of SsPG1 during infection is consistent with the finding of a previous study[51]in which SsPG1 was identified as the most highly expressed gene among the four endo-PG and two exo-PG genes during infection.Amselem et al.[41] pointed out that the S.sclerotiorum genome is especially well suited for pectin decomposition and that the fungus grows well on pectin and poorly on xylan and cellulose.The evidence cited above suggests that PG is one of the most important CWDEs in S.sclerotiorum pathogenicity.Furthermore,of all the pathogenic factors of S.sclerotiorum,OA has received the most attention,and SsOAH1 is essential for S.sclerotiorum OA biogenesis.Accordingly,in this study,the pathogenicity genes SsPG1,SsOAH1 and SsCBH were selected as potential targets for HIGS.

Koch et al.[22] found that in vitro treatment of the fungal pathogen F.graminearum with dsRNAs targeting CYP51 genes silenced the expression of one or more CYP51 genes in the developing fungus.Wang et al.[27] found that B.cinerea could take up fluorescein-labeled dsRNAs and sRNAs from the environment,which then induced gene silencing in fungal cells,a phenomenon termed environmental RNAi.In agreement with the findings of McLoughlin et al.[35],we found that co-incubation of dsRNAs with S.sclerotiorum in axenic liquid medium reduced the transcript levels of target genes(Fig.1D–F).These results suggest that S.sclerotiorum takes up dsRNAs from the environment and processes them with its own RNAi machinery.

After verifying environmental RNAi activity in S.sclerotiorum,we also found that the application of dsRNAs targeting pathogenicity-associated genes on plant surfaces conferred protection against S.sclerotiorum (Fig.2).Similarly,external application of synthetic sRNAs or dsRNAs to the plant surface (SIGS) has been shown to protect against multiple pathogens,such as B.cinerea[27],F.graminearum [23,30],and Phakopsora pachyrhizi [26].The present study not only indicates the substantial potential of using SIGS-based fungicides to control Sclerotinia rot but also shows that silencing the SsPG1,SsCBH,and SsOAH1 genes can inhibit S.sclerotiorum infection,furthering the selection of efficient HIGS targets.

By application of environmental RNAi and SIGS in S.sclerotiorum,we performed HIGS by stably expressing hairpin RNAs complementary to the genes SsPG1,SsCBH,and SsOAH1 of S.sclerotiorum in B.napus.The presence of target gene-specific siRNAs in HIGS transgenic plants was demonstrated by small-RNA sequencing(Figs.3,6B),indicating their successful expression.This result further confirmed that the target gene silencing observed in S.sclerotiorum-inoculated transgenic leaves was due to the presence of gene-specific siRNAs.HIGS conferred B.napus resistance to S.sclerotiorum in B.napus tissues at several developmental stages(Figs.4,5),indicating that HIGS can provide long-lasting protection.Inheritance of the resistance confirmed its genetic stability(Fig.4).The off-targets effects of the HIGS constructs in B.napus were further confirmed by investigating the agronomic traits in the T2generation (Table S2).Although the SsPG1,SsCBH,and SsOAH1 genes are important for the pathogenicity of S.sclerotiorum,the disease lesion areas on the leaves were reduced at 48 hpi by 20.8%–38.7%in single-target HIGS lines(Fig.4).To increase the pathogen inhibition,we designed a chimeric hairpin RNAi construct to simultaneously silence SsPG1,SsCBH,and SsOAH1 and found that the triple-target HIGS lines showed stronger protection phenotypes than the single-target HIGS lines.

Fig.6.Simultaneous silencing of SsPG1,SsOAH1,and SsCBH in S.sclerotiorum by HIGS conferred increased resistance.(A) Schematic representation of the triple-target HIGS construct.A 750-bp tandem DNA fragment containing three 250-bp DNA fragments from SsPG1,SsOAH1,and SsCBH was inserted into the pMDC83-ihpRNAi vector in sense and antisense orientations.(B)Small RNA profiling of S.sclerotiorum target genes in uninoculated leaves of triple-target HIGS transgenic plants(T1).The coverage depth and alignment track were obtained by mapping the Illumina sequence reads to the 750-bp tandem DNA sequence.(C) Expression levels of target genes in the S.sclerotioruminfected J9712 line and the triple-target-RNAi-4 line at 24 hpi.Values were normalized to the fungal endogenous control gene Sstub1 (SS1G_04652).Error bars indicate standard deviations of three replicates.(D–G) Assessment of the disease resistance of triple-target HIGS transgenic B.napus (T1) to S.sclerotiorum by the detached-leaf inoculation(D,E)and cotyledon inoculation(F,G)methods.Error bars represent the standard error of the lesion sizes from 60 leaves and cotyledons of each line(20 leaves and cotyledons for each of the three replicates).Significance compared with J9712 (*, P ＜0.05;**, P ＜0.01,Student’s t-test).Scale bars,1 cm in (E) and (G).

Thus,B.napus can export heterogenous expressed sRNAs that are complementary to pathogenicity-associated genes of S.sclerotiorum.Our failure to observe complete resistance to S.sclerotiorum in the HIGS lines may have been due to the presence of residual transcripts of target genes still conferring pathogenicity.The pathogenicity of S.sclerotiorum is complex and involves multiple factors.For example,OA has been considered a pathogenicity factor essential for S.sclerotiorum infection since 1990,when a UV mutant that was deficient in OA production was developed [37].In a recent study [48],mutation of SsOAH1 led to complete loss of OA production and did not prevent establishment of a primary lesion,but did prevent lesion spread and associated host colonization.In recent studies [52,53],host plants secreted exosome-like extracellular vesicles to deliver endogenous plant sRNAs to fungal pathogens and inhibit fungal virulence genes,suggesting that HIGS is also a natural defense mechanism in plants[54].Host cells transfer endogenous sRNAs into fungal cells not simply via concentration-dependent diffusion but rather through a selective process [53,55].Thus,selective sRNA secretion could be another factor affecting HIGS efficiency.Future studies should focus on improving HIGS efficacy and exploiting the most efficient HIGS target.

We conclude that SIGS and HIGS of key pathogenic genes are effective strategies for controlling Sclerotinia rot.Simultaneously silencing multiple S.sclerotiorum pathogenic genes by HIGS conferred increased resistance to S.sclerotiorum in B.napus.To our knowledge,this is the first report of suppression of pathogen infection in Brassica species using HIGS.

Declaration of competing interest

The authors declare no conflicts of interest.

CRediT authorship contribution statement

Jian Wu:Supervision,Writing–original draft,Funding acquisition.Shengliang Yin:Visualization,Investigation.Li Lin:Investigation.Dongxiao Liu:Investigation.Sichao Ren:Investigation.Wenjing Zhang:Investigation.Wencheng Meng:Validation,Data curation.Peipei Chen:Validation,Data curation.Qinfu Sun:Resources.Yujie Fang:Project administration.Cunxu Wei:Resources.Youping Wang:Writing– review &editing,Supervision,Funding acquisition.

Acknowledgments

This work was supported by the National Natural Science Foundation of China(32072020,U20A2028,and 31901504),the Jiangsu Agricultural Science and Technology Innovation Fund (CX(20)3120),the Project of Special Funding for Crop Science Discipline Development (yzuxk202006),the Priority Academic Program Development of Jiangsu Higher Education Institutions and the Qinglan Project of Yangzhou University.

Appendix A.Supplementary data

Supplementary data for this article can be found online at https://doi.org/10.1016/j.cj.2021.08.007.