circRNAs Are Involved in the Rice-Magnaportheoryzae Interaction1[OPEN]

Jing Fan,a,2 Weili Quan,b,2 Guo-Bang Li,a,2 Xiao-Hong Hu,a Qi Wang,b He Wang,a Xu-Pu Li,a Xiaotian Luo,c

Qin Feng,a Zi-Jin Hu,a Hui Feng,a Mei Pu,a Ji-Qun Zhao,a Yan-Yan Huang,a Yan Li,a Yi Zhang,b andWen-Ming Wanga,3,4

aState Key Laboratory of Crop Gene Exploration and Utilization in Southwest China, Rice Research Institute,Sichuan Agricultural University, Chengdu 611130, Sichuan, ChinabCenter for Genome Analysis, ABLife Inc., Wuhan 430075, Hubei, ChinacLaboratory for Genome Regulation and Human Health, ABLife Inc., Wuhan 430075, Hubei, China

ORCID IDs: 0000-0002-6747-4302 (J.F.); 0000-0003-4066-4875 (W.-M.W.).

Circular RNAs (circRNAs) play roles in various biological processes, but their functions in the rice (Oryza sativa) response toMagnaporthe oryzae remain elusive. Here, we demonstrate that circRNAs are involved in the rice-M. oryzae interaction usingcomparative circRNA-sequencing and transgenic approaches. We identified 2932 high-confidence circRNAs from young leavesof the blast-resistant accession International Rice Blast Line Pyricularia-Kanto51-m-Tsuyuake (IR25) and the blast-susceptibleaccession Lijiangxin Tuan Heigu (LTH) under M. oryzae-infected or uninfected conditions; 636 were detected specifically uponM. oryzae infection. The circRNAs in IR25 were significantly more diverse than those in LTH, especially under M. oryzaeinfection. Particularly, the number of circRNAs generated per parent gene was much higher in IR25 than in LTH andincreased in IR25 but decreased in LTH upon M. oryzae infection. The higher diversity of circRNAs in IR25 was furtherassociated with more frequent 39 and 59 alternative back-splicing and usage of complex splice sites. Moreover, a subset ofcircRNAs was differentially responsive to M. oryzae in IR25 and LTH. We further confirmed that circR5g05160 promotes riceimmunity against M. oryzae. Therefore, our data indicate that circRNA diversity is associated with different responses toM. oryzae infection in rice and provide a starting point to investigate a new layer of regulation in the rice-M. oryzae interaction.

Rice (Oryza sativa) is a staple crop that feeds morethan half of the global population (Liu et al., 2014). Riceblast, caused by the fungal pathogen Magnaportheoryzae, is one of the most devastating diseases threat-ening rice productionworldwide (Liu andWang, 2016).Rice has multiple layers of defense against the invasionof blast fungus (Liu et al., 2013). First, several ricepattern-recognition receptors, including Chitin Elici-tor Binding Protein (CEBiP), Chitin Elicitor ReceptorKinase1 (CERK1), Lysin Motif-Containing Protein4

(LYP4), and LYP6, recognize chitin fragments of theblast fungus and trigger defense responses (Shimizuet al., 2010; Liu et al., 2012) called pathogen-associatedmolecular pattern-triggered immunity (PTI). PTI can besuppressed by effector proteins secreted by M. oryzae(Khang et al., 2010; Mentlak et al., 2012). To counteractthe pathogen, rice deploys resistance (R) proteins torecognize avirulence effectors from M. oryzae, leadingto a second layer of defense called effector-triggeredimmunity (ETI). More than 100 R genes have beenidentified, and about 30 of them have been functionallycharacterized to act as on-off switches in regulating riceblast resistance (Wang et al., 2017a). Some of the Rgenes, such as Pigm and Pi2, have been widely exploi-ted in blast disease-resistance programs (Shi et al., 2015;Deng et al., 2017). Both PTI and ETI can be modulatedby microRNAs (miRNAs; Padmanabhan et al., 2009).

miRNAs are a class of noncoding RNAs that actin various biological processes and stress-induced re-sponses. Increasing evidence supports the role ofmiRNAs in fine-tuning rice immunity againstM. oryzae.Genome-wide small RNA analyses have revealed anumber of candidate miRNAs responsive to M. oryzaeinfection or elicitors (Campo et al., 2013; Li et al., 2014;Li et al., 2016;Wang et al., 2018). Transgenic approachesfurther confirm miRNAs, such as miR7695, miR398b,miR160a, andmiR166k-166h (Campo et al., 2013; Li et al.,2014, 2019; Salvador-Guirao et al., 2018), positively

1This work was supported by the grants from the National NaturalScience Foundation of China (31430072, 31672090, 31772241) and agrant from ABLife Inc. (ABL2015-01015).

2These authors contributed equally to the article.3Author for contact: j316wenmingwang@sicau.edu.cn4Senior author.The author responsible for distribution of materials integral to the

findings presented in this article in accordance with the policy de-scribed in the Instructions for Authors (www.plantphysiol.org) is:Wen-Ming Wang (j316wenmingwang@sicau.edu.cn).

J.F., W.Q., Y.Z., and W.-M.W. designed the research; J.F., W.Q.,G.-B.L., X.-H.H., Q.W., H.W., X.-P.L., X.L., Q.F., Z.-J.H., H.F, M.P.,J.-Q.Z., and Y.L. performed the experiments; J.F., W.Q., G.-B.L., Q.W.,Y.Z., and W.-M.W. analyzed and interpreted the data; J.F., W.Q.,G.-B.L., Q.W., Y.Z., andW.-M.W. wrote the manuscript. J.F., W.Q., andG.-B.L. contributed equally to this work.

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regulate rice blast resistance; on the contrary, miR169a,miR164a, miR319b, miR396, andmiR167d (Li et al., 2017;Wang et al., 2018; Zhang et al., 2018; Chandran et al.,2019; Zhao et al., 2019) act as negative regulators of riceimmunity against the blast pathogen. miRNAs exerttheir biological functions via targeting mRNAs with se-quence complementarity (Seitz, 2009); meanwhile,binding of miRNAs to their targets could be interferedwith by endogenous noncoding RNAs, such as targetmimics (Wu et al., 2013) and circular RNAs (circRNAs;Hansen et al., 2013). Target mimics have been reportedto regulate rice immunity against the blast pathogen(Li et al., 2017; Chandran et al., 2019; Li et al., 2019), but itis unknown whether circRNAs are involved in the rice-M. oryzae interaction.circRNAs are produced from precursor messenger

RNAs through back-splicing in which an upstream 39splicing acceptor site is joined to a downstream 59splicing donor site (Ashwal-Fluss et al., 2014). Accord-ing to their genomic origins, circRNAs are mainlyclassified as exonic, intronic, exon-intronic, and inter-genic (Bolha et al., 2017). circRNAs regulate gene ex-pression in animals through different mechanisms,such as miRNA sponges, specific RNA-RNA interac-tions, and affecting alternative splicing (Li et al., 2018).Since the identification of their roles in regulating geneexpression in animals (Memczak et al., 2013), circRNAshave also been identified in different plant species, in-cluding Arabidopsis (Arabidopsis thaliana), rice, wheat(Triticum aestivum), barley (Hordeum vulgare), maize(Zea mays), tomato (Solanum lycopersicum), potato (So-lanum tuberosum), soybean (Glycine max), cotton (Gos-sypium hirsutum), and kiwifruit (Actinidia deliciosa;Wang et al., 2014, 2017b, 2017c; Lu et al., 2015; Yeet al., 2015, 2017; Darbani et al., 2016; Zuo et al., 2016;Chen et al., 2017, 2018; Zhao et al., 2017).While reports identifying plant circRNAs are rapidly

increasing, investigation of their roles is far more chal-lenging. Plant circRNAs have been found to functionduring fiber development, flowering, and fruit colora-tion based on spatial- and/or tissue-specific expressionpatterns (Tan et al., 2017; Wang et al., 2017c; Tong et al.,2018). The expression of plant circRNAs also respondsto abiotic and biotic stimuli, including pathogen inva-sion (Zuo et al., 2016; Wang et al., 2017c; Zhou et al.,2017; Ghorbani et al., 2018; Xiang et al., 2018; Gao et al.,2019). For example, the accumulation of a set of circR-NAs is altered in kiwifruit upon canker pathogen in-fection (Wang et al., 2017c). However, it is unknownwhether circRNA production and expression changesare functionally linked to plant disease resistance.To explore whether circRNAs are involved in rice

blast resistance, we performed genome-wide identifi-cation of circRNAs in leaf transcriptomes of the resis-tant accession International Rice Blast Line PyriculariaKanto51-m-Tsuyuake (designated as IR25) and theblast-susceptible accession Lijiangxin Tuan Heigu(LTH) before and after M. oryzae infection via the ri-bosomal RNA-depleted RNA-sequencing technique(Lu et al., 2015). circRNAs were identified with the

CIRI2 pipeline, and their numbers and abundance werecompared between IR25 and LTH, and/or between thesamples during M. oryzae infection and without infec-tion. circRNAs in IR25 were more diverse than those inLTH regardless of M. oryzae infection, which was as-sociated with the increased complexity of alternativesplicing sites. We also obtained transgenic lines over-expressing circR5g05160 and confirmed that up-regulationof this circRNA led to enhanced blast disease resistance.Overall, our results indicate that increased alternativesplicing complexity contributes to the diversity ofcircRNAs, and circRNAs are involved in the rice-M. oryzae interaction.

RESULTS

Identification of High-Confidence circRNAs from the LeafTranscriptomes of Blast-Resistant and Blast-SusceptibleRice Accessions

To explore the different responses to M. oryzae be-tween blast-resistant and blast-susceptible rice acces-sions, we performed RNA-sequencing (RNA-seq) onthe leaves from 3-week-old seedlings of IR25 and LTHbefore (IR25-0h, LTH-0h) and after M. oryzae infection(IR25-12h, LTH-12h, IR25-24h, LTH-24h). In total, weobtained 18 transcriptome data sets with each con-taining an average of about 200 million paired-endreads for analysis (Supplemental Table S1). Then, weperformed principle component analysis based on theexpression level of each gene and found a clear sepa-ration of both the LTH and IR25 rice samples betweenbefore (0 h) and afterM. oryzae infection (12 h and 24 h)in two experiment benches (Supplemental Fig. S1A).Samples from bench 1 and bench 2 were also obviouslyseparated (Supplemental Fig. S1A). However, therewas no clear separation between LTH and IR25 samplesat each time point from either experimental bench(Supplemental Fig. S1A). These results indicated theoverall gene expression profiles were similar betweenIR25 and LTH, regardless of M. oryzae infection.Next, we performed edgeR analysis to identify dif-

ferentially expressed genes (DEGs; $2-fold increase ordecrease, false discovery rate [FDR], 0.05) between thetwo accessions at each time point. From one bench, atotal of 914, 1456, and 1758 DEGs were found in IR25compared to the LTH at 0, 12, and 24 h, respectively(Supplemental Fig. S1B, left). From the other bench, thenumber of DEGs were 4352, 2687, and 2855 at 0, 12, and24 h, respectively (Supplemental Fig. S1B, right). Forexpression levels of all unique DEGs, unsupervisedhierarchical clustering analysis revealed a clear differ-ence between LTH and IR25 rice samples in response toM. oryzae infection (Supplemental Fig. S1C). As to theup- and down-regulated DEGs between IR25 and LTH,gene ontology (GO) analysis uncovered a consistentlysignificant enrichment in “response to stress” and “re-sponse to biotic stimulus” (Supplemental Fig. S2).These data indicate that the transcriptomes of IR25 and

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LTH are differently modulated in response to infectionwithM. oryzae and thus are suitable to screen circRNAsdifferentially responsive to M. oryzae.

To identify circRNAs from leaf transcriptome data ofboth IR25 and LTH, we removed low-quality reads,and the rest of the reads were run with a custompipeline based on CIRI2, followed by a validationmapping step (Supplemental Fig. S1D, left). In thevalidation step, we aligned all reads onto an artificialcircRNA sequence database built for all circRNAs re-trieved from the CIRI2 pipeline, and only circRNAswith at least two mapped reads covering the back-splicing junction were kept as candidate circRNAs(Supplemental Fig. S1D, right). A total of 374 to 1527circRNAs were identified in each of the IR25 and LTHtranscriptome samples (Fig. 1A). Overall, 2932 circR-NAs were identified in all samples, of which 72.74%,22.21%, and 5.05%were exonic, intergenic, and introniccircRNAs, respectively (Supplemental Table S2). ThesecircRNAs were distributed across the whole genomeand were more commonly found at both ends of chro-mosomes (Fig. 1B). Such distribution is similar to a re-cent report in maize (Chen et al., 2018). A total of 1646circRNAs were previously reported and deposited inPlantcircBase for rice (release 3; Chu et al., 2017), while1286 circRNAs were only detected in this study(Supplemental Table S2). Moreover, 636 circRNAswere specifically triggered by M. oryzae infection(Supplemental Table S3). Out of them, 411 and 144circRNAs were detected only inM. oryzae-infected IR25and LTH, respectively; 81 circRNAs were common inboth M. oryzae-infected IR25 and LTH (SupplementalTable S3). Kyoto Encyclopedia of Genes and Genomes(KEGG) enrichment analysis indicated that the parentgenes producing M. oryzae-triggered circRNAs wereinvolved in multiple stress-responsive pathways,such as biosynthesis of secondary metabolites, pro-tein export, and terpenoid backbone biosynthesis(Supplemental Table S4). These results indicate thatcircRNAs widely exist in leaf transcriptomes of bothIR25 and LTH and M. oryzae infection induces moreproduction of circRNAs in IR25 than in LTH.

To validate these circRNAs, we randomly selected 12circRNAs and validated them by PCR using pairs ofdivergent and convergent primers (Supplemental TableS5). All 12 pairs of convergent primers successfullyamplified the expected length of fragments from bothcDNAs of total RNAs and genomic DNA (Fig. 1C,Supplemental Fig. S3A). By contrast, the divergentprimers could only amplify fragments from cDNA oftotal RNAs, but not from genomic DNA (Fig. 1C;Supplemental Fig. S3A). The amplification pro-ducts from the divergent primers were confirmedto span the back-splicing junction of circRNAs viaSanger sequencing and sequence mapping (Fig. 1D;Supplemental Fig. S3B). Moreover, all 12 pairs of di-vergent primers yielded amplification products fromcDNAs of total RNAs with or without treatment ofRNase R that digests linear RNAs (Fig. 1E). These dataindicate the validation of all 12 randomly selected

circRNAs. Thus, the circRNAs identified by our anal-ysis pipeline are highly reliable.

The Diversity of circRNAs Is Constantly Higher in IR25Than in LTH Regardless of M. oryzae Infection

We noticed more circRNAs were repeatedly identi-fied in IR25 than in LTH (Fig. 1A). This prompted us todetermine the difference between blast-resistant andsusceptible rice accessions in producing circRNAs. Atotal of 2633 and 1787 circRNAswere identified from allsamples of IR25 and LTH, respectively, of which 1488circRNAs overlapped (Fig. 2A, top), indicating most ofthe LTH-produced circRNAs are included in those ofIR25. Focusing on the circRNAs detected in at least twosamples, 1204 and 688 circRNAs remained in IR25 andLTH, respectively, of which 588 overlapped (Fig. 2A,bottom), indicating more diverse circRNAs in IR25than in LTH. To remove the potential effect of se-quencing bias on the number of identified circRNAs,we obtained the normalized number of circRNAs ineach million of high-quality sequencing tags (Fig. 2B),which confirmed IR25 generated more circRNAs thandid LTH.

To explore whether the increased circRNA diversityin IR25 depended on theM. oryzae infection, we plottedthe normalized number of circRNAs to each postin-fection time point. The results demonstrated thatM. oryzae infection led to a marginal increase in thecircRNA diversity; however, the circRNA diversity inIR25 was constantly higher than that in LTH (Fig. 2C).We then analyzed circRNAs located in the gene regionsand found that the diversity increase became morepronounced in IR25 but not in LTH (Fig. 2D), indicatingthe biogenesis of diverse circRNAs from genic locimight be specifically triggered in the blast-resistantaccession by the M. oryzae infection.

To further dissect the potential origins of the in-creased circRNA diversity in IR25, we analyzed thenumber of circRNAs generated by each gene. The av-erage number of circRNAs per gene was constantlyhigher in IR25 than in LTH (Fig. 2E), implying genes inIR25 are more capable of generating circRNAs thanthose in LTH. Moreover, the numbers of circRNA-generating genes were 275 in common, 360 specifi-cally in IR25, and 57 specifically in LTH (Fig. 2F;Supplemental Table S6), indicating there were morecircRNA-generating genes in IR25 than in LTH, andmost (;82.9%) of the circRNA-generating genes in LTHwere included in IR25. Then, we analyzed the averagenumber of circRNAs generated by each gene of the275 circRNA-producing genes shared by IR25 and LTH.The average number of circRNAs generated per genewas significantly higher in IR25 than in LTH (Fig. 2G).Upon infection withM. oryzae, the number of circRNAsgenerated per gene was obviously reduced in LTH; bycontrast, it was increased in IR25, and the increase be-came significant at 24 h post inoculation (hpi) withM. oryzae (Fig. 2G). Taken together, our results indicate

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Figure 1. Genome-wide identification of circRNAs in leaves of the blast-resistant accession International Rice Blast Line Pyr-icularia Kanto51-m-Tsuyuake (IR25) and the blast-susceptible accession Lijiangxin Tuan Heigu (LTH). A, Number of identified

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that IR25 is capable of producing more circRNAs thanLTH, particularly upon M. oryzae infection.

It has been reported that circRNAs can regulate theexpression of their parent genes, therefore exerting abiological function associated with their parent genes(Li et al., 2018). To uncover whether the circRNA-generating genes possess any blast resistance func-tions, we subjected the circRNA-generating genes tofunctional enrichment analysis according to the circR-NAs specifically detected in IR25 and LTH and thoseshared by IR25 and LTH. We found regulation of au-tophagy and peroxisome were among the top func-tional terms enriched by circRNA-generating genes andwere particularly more enriched by IR25-specific genes(Fig. 2H; Supplemental Fig. S4). As autophagy andperoxisomes have been documented to function inplant immunity (Mammarella et al., 2015; Hofius et al.,2017), our findings imply a link between circRNAs andblast resistance.

Alternative Splicing Contributes to the Increased circRNADiversity in IR25

Since alternative back splicing is required for the bi-ogenesis and diversification of circRNAs in rice (Luet al., 2015; Ye et al., 2017), we hypothesized more di-verse circRNAs in IR25 may be attributed to morecomplicated alternative splicing in IR25 than in LTH.Therefore, we annotated the downstream 59 back-splicesites and upstream 39 back-splice sites from all theidentified circRNAs. As expected, the numbers of bothalternative 59 back-splice sites and 39 back-splice siteswere significantly higher in IR25 than in LTH regard-less of M. oryzae infection (Fig. 3, A and B). We furtherviewed the alternative back-splicing events by calcu-lating their percent circularized-site usage (PCU); eachisoform of an alternative back-splicing event had itsown PCU (Supplemental Table S7). The heatmap plot ofPCUs showed alternative back-splicing events weremore prevalent in IR25 samples than in LTH samples(Fig. 3C). These results indicated alternative back-splicing patterns were more diverse in IR25 than inLTH, which could contribute to the increased circRNAdiversity in IR25.

Internal alternative splicing inside circRNAs is alsoessential for the biogenesis and diversity of circRNAs inanimals (Gao et al., 2016; Zhang et al., 2016). A numberof rice circRNAs consist of multiple exons that couldbe derived from internal splicing events of circRNAs

(Ye et al., 2017). Therefore, we hypothesized that morediverse circRNAs in IR25 may also be attributed tomore internal alternative splicing in circRNAs of IR25than those of LTH. To this end, we adopted CircRNAIdentifier-Alternative Splicing (CIRI-AS; Gao et al.,2016) to identify and analyze the internal alternativesplicing in circRNA. Indeed, internal alternative splic-ing events inside circRNAs were significantly moreprevalent in IR25 than in LTH, although all alternativesplicing events were not obviously different betweenIR25 and LTH (Fig. 3D). In addition, all four basictypes of alternative splicing were higher in IR25 thanin LTH (Fig. 3E).

Taken together, our results demonstrated both al-ternative back-splicing and internal alternative splicinginside circRNAs may contribute to the higher diversityof circRNAs in IR25 than in LTH.

circRNAs Are More Likely Derived from ComplexSplice Sites

Recent studies in mammals have shown the presenceof very complex splice sites that generate splice junc-tions with more than two donors or acceptors (Gaoet al., 2016; Zhang et al., 2016; Feng et al., 2019; Zhenget al., 2019). We speculated complex splice sites shouldhave a higher probability of successful back-splicing.To test this hypothesis, we analyzed the number ofsplice junctions formed by each detected splice site in allsamples. The number of splice junctions displayedsimilar profiles in both IR25 and LTH (SupplementalFig. S5). Most splice sites underwent only one splicingevent, which yielded one splice junction. Nevertheless,a significant fraction of splice sites underwent two orthree splicing events, and thousands of splice sites un-derwent four or more splicing events (SupplementalFig. S5).

We then analyzed whether the linear and circRNAsplice sites were associated with their complexity. Thetotal number of circRNA splice sites was less than thatof the linear splice sites (Fig. 4A, left). However, thepercentage of complex splice sites ($2 junctions) incircRNAs was more than that in linear transcripts; bycontrast, the percentage of simple splice sites (junc-tion 5 2) in circRNAs was significantly lower than thatin linear transcripts (Fig. 4A, right). We further ex-plored whether the association between circRNAs, andtheir splice site complexity was different in IR25 andLTH. Both the number (Fig. 4B, left) and percentage

Figure 1. (Continued.)circRNAs in each sample from two independent experimental benches. Rep 1 and Rep 2 for each sample is two independentbiological replicates that were sequenced in one bench experiment. Rep 3 is amixture of total RNAs fromRep 1 and Rep 2 of eachsample in the other bench experiment. B, Distribution of back-splicing reads of circRNAs in each chromosome. Distribution ofmRNA reads in LTH_24h was randomly selected as a control. C, Representative circRNAs validated by PCR amplification withdivergent and convergent primers. D, Representative circRNAs validated by Sanger sequencing. E, Relative expression of theindicated representative circRNAs in IR25 and LTH detected by RT-qPCR after RNase R treatments. R1, R2 represent sampleswith and without RNase R treatment, respectively. Error bars indicate SD (n 5 3).

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(Fig. 4B, right) of complex splice-site-associatedcircRNAs were higher in IR25 than in LTH, al-though the differences were not significant. UsingLOC_Os11g36030 and LOC_Os2g148000 as examples,the reads distribution demonstrated diverse circRNAswere generated from more complicated splice sites inIR25 than in LTH (Fig. 4C). These results indicate IR25has a high capacity for supporting complex splicing,which may facilitate back-splicing and circRNAproduction.

The Abundance of Some circRNAs Is Significantly Alteredupon M. oryzae Infection

To explorewhether circRNA abundance responded toM. oryzae infection, we violin-plotted circRNA expres-sion level (total circRNA junction reads per mapped

million reads). Total circRNA abundance was margin-ally different among samples (Supplemental Fig. S6).We then screened circRNAs differentially expressed inIR25 and LTH in response to M. oryzae infection andidentified 20 and 31 up-regulated circRNAs and eightand 16 down-regulated circRNAs in IR25 at 12 and 24hpi, respectively. By contrast, we identified six and fiveup-regulated circRNAs and 21 and 13 down-regulatedcircRNAs in LTH at 12 and 24 hpi, respectively (Fig. 5A;Supplemental Table S8). Apparently, the number ofup-regulated circRNAs was more than that of down-regulated circRNAs in IR25 uponM. oryzae infection; onthe contrary, more circRNAs tended to be down-regulated in LTH (Fig. 5A). There were 18 circRNAsconstantly up-regulated and six circRNAs constantlydown-regulated at both 12 and 24 hpi in IR25, and twocircRNAs constantly up-regulated and eight circRNAsconstantly down-regulated in LTH (Fig. 5B). These

Figure 2. circRNAs aremore diverse in IR25 than in LTH. A, Number of overlapped circRNAs between IR25 and LTH identified inat least one sample of IR25 and LTH (top). Number of overlapped circRNAs identified in at least two repeated samples of IR25 andLTH (bottom). B, The normalized numbers of circRNAs in each million of high-quality sequencing tags in IR25 and LTH. C, Thenumber of circRNAs normalized to each million of high-quality sequencing tags in IR25 and LTH at 0, 12, and 24 h postinoc-ulation (hpi) ofM. oryzae. D, The number of circRNAs in the gene regions normalized to eachmillion of high-quality sequencingtags in IR25 and LTH at 0, 12, and 24 hpi ofM. oryzae. E, The average number of per-gene-derived circRNAs detected in at leastone sample of IR25 and LTH at 0, 12, and 24 hpi ofM. oryzae. F, Venn diagram analysis of the number of parent genes generatingcircRNAs identified in at least two repeated samples of IR25 and LTH. G, The average number of per-gene-derived circRNAsidentified in at least two replicated samples of IR25 and LTH at 0, 12, and 24 hpi ofM. oryzae. The circRNAs from the overlappedparent genes in F were analyzed. H, KEGG analysis of host genes producing IR25-specific circRNAs (IR25 uniq). Error bars in B toE and G indicate SD (n 5 3). Asterisks indicate significant differences detected by Student’s t test (*P , 0.05, **P , 0.01, and***P , 0.001).

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circRNAs could be the ones involved in rice responseto M. oryzae. As an example, the expression of acircRNA from LOC_Os12g19381 was constantly down-regulated upon M. oryzae infection in both IR25 andLTH (Fig. 5C). LOC_Os12g19381 was annotated withthe putative function of ribulose bisphosphate carbox-ylase, implying M. oryzae infection might affect ricephotosynthesis.

Next, we screened circRNAs differentially expressedbetween IR25 and LTH. We identified 38, 40, and 51circRNAs with higher expression and 15, 12, and 14circRNAs with lower expression in IR25 than in LTHat 0, 12, and 24 hpi, respectively (Fig. 5D). ThesecircRNAs may be associated with regulation of blastdisease resistance. Moreover, a significant number ofcircRNAs overlapped among different time points(Fig. 5E; P , 0.05). As an example, a circRNA fromLOC_Os11g11890 was shown to have constantly

higher expression in IR25 than in LTH (Fig. 5F).LOC_Os11g11890 was annotated as an ortholog of thedisease-resistance gene RPG1 in barley (Brueggemanet al., 2002), suggesting IR25 might deploy circRNAsfor blast resistance.

To validate these differentially expressed circRNAs,we randomly selected 12 circRNAs for reverse tran-scription quantitative PCR (RT-qPCR). All 12 circR-NAs were differentially expressed after M. oryzaeinfection in both IR25 and LTH, which was consistentwith the circRNA-seq results (Fig. 5G; SupplementalFig. S7). These results demonstrated the abundance ofmost rice circRNAs was higher in IR25 than in LTH,and infection of M. oryzae led to more up-regulatedcircRNAs in IR25 but more down-regulated circR-NAs in LTH. Therefore, the differentially expressedcircRNAs should be a priority for functional charac-terization in the future.

Figure 3. Alternative splicing patterns of circRNAs were more diverse in IR25 than in LTH. A and B, The normalized number ofalternative 39 back-splice sites (A) and 59 back-splice sites (B) per million mapped reads in each sample of IR25 and LTH at 0, 12,and 24 h postinoculation (hpi) ofM. oryzae. C, Heatmap of the percent circularized-site usage (PCU) of the use of proximal anddistal 59 back-splice sites (or 39 back-splice sites) in each sample. PCUs were quantified as the number of detected back-splicereads for two junctions with common 59 or 39 back-splice sites using the formula: reads for back-splicing junction a /(reads forback-splicing junction a 1 reads for back-splicing junction b). D, All alternative splicing events detected (left) and alternativesplicing events of circRNAs (right) in IR25 and LTH at 0, 12, and 24 hpi ofM. oryzae. E, Numbers of four basic types of alternativesplicing events in circRNAs identified in each sample of IR25 and LTH at 0, 12, and 24 hpi ofM. oryzae. A3SS, alternative 39 splicesite. A5SS, alternative 59 splice site. ES, Exon skipping; IntronR, intron retention. Error bars in A, B, and D indicate SD (n 5 3).Asterisks indicate significant differences detected by Student’s t test (*P , 0.05 and **P , 0.01).

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Overexpression of circR5g05160 Leads to EnhancedBlast Resistance

To confirm the involvement of circRNAs in reg-ulation of the rice-M. oryzae interaction, we se-lected a circRNA Chr5:2512798j2514806 derived fromLOC_Os05g05160 (circR5g05160) for functional analy-sis. We selected circR5g05160 because it was preferen-tially expressed in IR25 (Supplemental Table S6), andits parent gene encodes a putative plant immunity

regulator MPK14 (Tena et al., 2011); moreover, thepredicted circR5g05160 sequence contained potentialtargeting sites of miRNAs (Supplemental Fig. S8C).We confirmed by qPCR that circR5g05160 was differ-entially responsive to M. oryzae with much higher in-duction in IR25 than in LTH, especially at early infectionstages (Fig. 6A).We then validated this circRNAvia PCRwith divergent and convergent primers (SupplementalFig. S8A; Supplemental Table S5) and confirmed the

Figure 4. Complex back-splice sites were higher in IR25 than in LTH. A, The number (left) and percentage (right) of simple splicesites (junction5 2) and complex splice sites (junction. 2) detected in all 18 data sets. circRNA_site is back-splice site. Linear_siteis forward-splice site. B, The number (left) and percentage (right) of simple back-splice sites and complex back-splice sitesdetected in IR25 and LTH, respectively. C, Visualization and reads distribution of alternative back-splice site usage of circRNAs atthe LOC_Os11g36030 (left) and the LOC_Os02g14800 (right) loci in one replicate of each sample of IR25 and LTH at 0, 12, and24 h postinoculation of M. oryzae. The numbers at the arc lines indicate the number of junction reads.

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junction site by Sanger sequencing (SupplementalFig. S8B). We also amplified the full length ofcircR5g05160 using the divergent primers and foundcircR5g05160 is a 607 nucleotide exonic circRNA(Supplemental Fig. S8C). There was an alternativesplicing event within circR5g05160 resulting in a10-nucleotide shift upstream of the second exon(Supplemental Fig. S8C). Sequence analysis con-firmed one potential targeting site for osa-miR168-5p and one for osa-miR2103 (Supplemental Fig. S8,C and D).

To overexpress circR5g05160 in rice, we first clonedthe DNA fragment containing predicted circR5g05160and its endogenous flanking introns into the pCAM-BIA1300 vector under the control of the Cauliflowermosaic virus 35S promoter (Supplemental Fig. S8E). Wethen tested whether this construct (p35S-circR5g05160)could efficiently produce circR5g05160 in the Nicotianabenthamiana transient expression system by qPCRwith divergent primers and Sanger sequencing.circR5g05160 was highly expressed in N. benthamiana,and the junction site and full-length sequence were

Figure 5. The changes in abundance of circRNAs in IR25 and LTH upon M. oryzae infection. A, The number of differentiallyexpressed circRNAs uponM. oryzae infection in IR25 and LTH. Up-regulated and down-regulated circRNAs were identified byedgeR with P , 0.05 and fold change$ 1.5. B, Overlap of up-regulated (top) and down-regulated (bottom) circRNAs at 12 and24 h postinoculation (hpi) in IR25 and LTH. LOC_Os12g19381 was the representative parent gene locus producing the down-regulated circRNAs. C, The distribution of back-splicing reads of a down-regulated circRNA derived from the LOC_Os12g19381locus in one replicate of the indicated samples. The numbers below each arc line indicate back-splicing read number. D, Thenumber of differentially expressed circRNAs between IR25 and LTH at 0, 12, and 24 hpi of M. oryzae infection. E, Overlap ofhigher (top) and lower (bottom) expression of circRNAs in IR25 compared to LTH at 0, 12, and 24 hpi of M. oryzae. F, Thedistribution of back-splicing reads of a differentially expressed circRNA at the LOC_Os11g11890 locus in one replicate of theindicated samples. The numbers below each arc line indicate back-splicing read number. G, RT-qPCR validation of the differ-ential expression of the circRNAChr11_6600899_6602460 at the LOC_Os11g11890 locus at indicated time points ofM. oryzaeinfection. Expression of the circRNAwas measured by circRNA-seq (left) and RT-qPCR (right). OsUbi was used as the referencegene in RT-qPCR. Gray bars and black bars represent IR25 and LTH, respectively. Error bars indicate SD (n 5 3).

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exactly the same as in rice (Supplemental Fig. S8, C andF), indicating the construct p35S-circR5g05160 can ex-press the full length of circR5g05160.Next,we introducedp35S-circR5g05160 into the rice accession Nippon-bare (NPB) via Agrobacterium tumefaciens-mediatedtransformation and obtained multiple independenttransgenic rice lines that overexpressed circR5g05160(Fig. 6B). In these lines, linear transcript accumula-tion of LOC_Os05g05160 was not greatly changed(Fig. 6B). Subsequently, two transgenic lines wereused for blast disease assay. Both lines formedmuch smaller disease lesions and supported signifi-cantly less fungal growth of M. oryzae than did NPB(Fig. 6, C–E), indicating enhanced resistance to theblast disease.To understand how circR5g05160 is involved

in blast resistance, we examined the expression ofsome defense-related genes in two transgenic lines

overexpressing circR5g05160. PTI-related genes,such as NAC4, PBZ1, and PR10b in rice (Li et al.,2014), were expressed at higher levels in over-expressing lines OX20 and OX24 than in wild-typeNPB; upon M. oryzae infection, their expressionlevels were induced much higher in OX20 and OX24than in NPB, especially at 12 hpi (Fig. 6, F–H). ETI-related genes, such asHSP90, SGT1, and RAR1 (Thaoet al., 2007; Shirasu, 2009), also displayed differen-tial expression in OX20 and OX24 compared to NPB.The basal expression levels of HSP90, SGT1, andRAR1 were 10- to 80-fold higher in OX20 and OX24than in NPB, although their expression levels weremostly similar between overexpressing lines andNPB after M. oryzae infection (Fig. 6, I–K). Theseresults indicate that circR5g05160 may be involvedin both PTI and ETI to regulate rice immunityagainst M. oryzae.

Figure6. Overexpressionof circR5g05160enhances rice resistance againstM. oryzae. A, RT-qPCR analysis ofcircR5g05160 in IR25 and LTHleaves infected with M. oryzae at indi-cated time points. OsUbi was used asthe reference gene. The expression ofcircR5g05160 in LTH_0h was set asthe control. B, Expression analysis ofcircR5g05160OX transgenic plants.The expression levels of circR5g05160and its parent gene LOC_Os05g05160were determined by RT-qPCR usingOsUbi as the reference gene. The ex-pression of indicated genes in NPB wasset as the control. C, Disease symptomof circR5g05160OX lines inoculatedwith M. oryzae strain eGFP-taggedZhong8-10-14 (GZ8). The leaves werephotographed at 7 days postinocula-tion. Scale bar 5 1 cm. D and E,Quantification of lesion length (D) andrelative fungal biomass (E) for the in-oculated leaves of indicated lines. Fto K, Expression analysis of defense-related genes in leaves of indicatedcircR5g05160OX lines and NPB at in-dicated time points after spraying in-oculation of GZ8. OsUbi was used asthe reference gene. The expression ofindicated genes in NPB_0h was set asthe control. Error bars in A and B and Dto K indicate SD (n 5 3). Student’s t testwas performed to examine the signif-icance of differences between LTHand IR25 (A) or between NPB andcircR5g05160OX transgenic lines (Band D to K) at indicated time points.Asterisks indicate significant differ-ences (*P , 0.05, **P , 0.01, and***P , 0.001).

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DISCUSSION

circRNAs are widespread and diverse in both ani-mals and plants and have potential regulatory func-tions (Li et al., 2018). In plants, circRNAs are closelyassociated with development and stress-induced re-sponses (Lu et al., 2015; Ye et al., 2015, 2017). Here,we demonstrated that circRNAs are involved in therice-M. oryzae interaction. First, out of 2932 high-confidence circRNAs identified in this study, 636circRNAs were specifically generated upon M. oryzaeinfection (Supplemental Table S3). Second, morecircRNAs were produced in the blast-resistant acces-sion IR25 than in the blast-susceptible accession LTH,and circRNA diversity was significantly increased byM. oryzae infection in IR25 but not in LTH (Fig. 2;Supplemental Table S3). The higher capability ofcircRNA biogenesis in IR25 was further associated withmore alternative back-splicing, internal alternative splic-ing inside circRNAs, and usage of complex splice sites(Figs. 3 and 4). Third, a subset of circRNAs was differ-entially responsive toM. oryzae in IR25 and LTH (Fig. 5),and functional analysis showed that circR5g05160 con-tributed to rice immunity against M. oryzae (Fig. 6).

As a posttranscriptional process in eukaryotic orga-nisms, alternative splicing leads to production of mul-tiple, distinct functional transcripts and diverseproteins from a single gene (Black, 2000; Nilsen andGraveley, 2010). Both alternative back-splicing and al-ternative splicing inside circRNA are required for thebiogenesis and diversity of circRNAs in animals (Gaoet al., 2016; Zhang et al., 2016; Feng et al., 2019; Zhenget al., 2019). In fact, alternative circularization ofcircRNA is reported as a common feature in rice, whichresults in a set of circRNA isoforms from the same locus(Lu et al., 2015; Ye et al., 2017). In this study, we foundmore circRNA production was associated with more 59and 39 alternative back-splice sites and more internalalternative splice sites inside circRNAs in IR25 than inLTH (Fig. 3), implying that internal alternative splicing,as well as alternative back-splicing, may contribute todifferent responses to M. oryzae in rice.

Complex splice sites can generate splice junctionswith more than two donors or acceptors (Gao et al.,2016; Zhang et al., 2016; Feng et al., 2019; Zheng et al.,2019). As alternative back-splicing events compete withthe canonical splice site and alternative splice site,complex splice sites may have more chances to producecircRNAs. In this study, the percentage of complexsplice sites was significantly higher in circRNAs than inlinear transcripts (Fig. 4). Moreover, both the numberand percentage of complex splice sites were obviouslyhigher in IR25 than in LTH, which is positively associ-ated with the capability of producing circRNAs in riceaccessions (Fig. 4). Therefore, complex splice sites maybe involved in circRNA biogenesis in response toM. oryzae in rice.

We observed that M. oryzae infection could spe-cifically trigger the production of some circRNAsin rice, of which the parent genes were enriched in

defense-related pathways, such as “Biosynthesis ofsecondary metabolites” and “Terpenoid backbone bi-osynthesis” (Piasecka et al., 2015; Supplemental TableS4). Particularly, a number of circRNAs (411) werespecifically induced in the resistant accession IR25upon M. oryzae infection. Their parent genes were sig-nificantly enriched in the KEGG pathway “Splicesome”(Supplemental Table S4), implying splicing-relatedgenes were strongly modulated, likely contributing toincreased circRNA diversity in IR25. Interestingly, forcircRNAparent genes shared by IR25 and LTH, those inIR25 showed a higher ability to generate circRNAs thanthose in LTH, especially under M. oryzae infection(Fig. 2G). Moreover, IR25-specific parent genes weresignificantly enriched in peroxisome- and autophagy-related pathways (Fig. 2H), which are involved in plantimmunity and disease resistance (Hofius et al., 2009;Daudi et al., 2012). For instance, peroxidases PRX33 andPRX34 are required for apoplastic reactive oxygenspecies production and basal resistance to pathogens inArabidopsis (Daudi et al., 2012). Autophagic compo-nents such as ATG6 function in hypersensitive celldeath and plant immunity (Hofius et al., 2009; Yueet al., 2015). Taken together, our data support thatproduction of rice circRNAs is responsive to M. oryzaeinfection, and some circRNAs may be involved in riceimmunity against M. oryzae.

Numerous circRNAs have been identified bygenome-wide analysis in plants, but relatively few havebeen functionally characterized. For instance, an exoniccircRNA from Arabidopsis SEPALLATA3 causes floralhomeotic phenotypes via increasing the expression ofthe cognate exon-skipped alternative splicing isoform(Conn et al., 2017). A grape (Vitis vinifera) circRNA Vv-circATS1 enhances cold tolerance when ectopicallyexpressed in Arabidopsis (Gao et al., 2019). The ricecircRNA Os08circ16564 was successfully overex-pressed in rice accession NPB, but the resulting trans-genic plants displayed no obvious phenotypes, and thebiological function of Os08circ16564 is still unknown(Lu et al., 2015). In this study, we demonstrated thefunction of circR5g05160 in rice resistance to blast dis-ease (Fig. 6), providing the first line of evidence thatcircRNAs function in the rice-M. oryzae interaction. Inaddition, we found potential miRNA-targeting sites incircR5g05160 (Supplemental Fig. S8, C and D); whetherthis circRNA modulates rice immunity throughsponging miRNAs could be a future research focus.Alternatively, circR5g05160 may interfere with thefunction of its parent gene OsMPK14, which belongs toa gene family critical for plant immunity (Tena et al.,2011).

CONCLUSION

In summary, we have identified a much largernumber/diversity of expressed circRNAs in leaves ofblast-resistant IR25 than in that of blast-susceptibleLTH. The difference may be associated with different

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responses to M. oryzae and attributed to differencesin efficiency of alternative back-splicing and usage ofcomplex splice sites. We also demonstrated a stronglink between circRNA production and rice blast resis-tance by functional analysis. These results provide newinsights into circRNA biogenesis in rice and uncoverregulatory factors in rice immunity, which will help usto understand the complicated regulatory network inthe rice-M. oryzae interaction.

MATERIALS AND METHODS

Rice Accessions and Fungi

Rice (Oryza sativa) plants used in this study were the blast-susceptible ja-ponica accession LTH and a corresponding blast-resistant accession IR25. IR25 isa monogenic line containing the blast resistance gene Pikm that was introducedinto LTH by backcrossing with Tsuyuake (Tsunematsu et al., 2000). All riceplants were grown at 26°C 6 2°C and 70% relative humidity under 12 hlight:12 h darkness. Magnaporthe oryzae strains (NC-24, NC-34, B9, Zhong8-10-14, 97-95-1, F1, NC-14, B13, and E37) were cultured in complete media at 28°Cunder 12 h light:12 h darkness for 2 weeks. Spores were collected and adjustedto a concentration of 23 105 spore mL21. Equal volume of spore suspension foreach strain were mixed and sprayed onto 3-week-old seedlings of LTH andIR25. Leaves were collected at 0, 12, and 24 hpi, immediately frozen in liquidnitrogen, and stored at 280°C until use.

circRNA-seq Library Construction and Sequencing

Leaf tissues of both rice accessions from the following three stages werecollected for library construction: before inoculation (0 h) and after inoculation(12 and 24 h). Total RNA of each sample was isolated using TRIzol (Ambion)according to the manufacturer’s instructions. To remove the noise from dif-ferences in individual plants and experimental variation, we generated a total ofthree sets of RNA-seq data from two independent benches. In one experimentalbench, we mixed total RNAs from different plants of two biological repeats forlibrary preparation. In the other bench, we used total RNAs from each bio-logical repeat for library construction (Supplemental Table S1).

Total RNA was treated with RQ1 DNase (Promega) to remove DNA. Thequality and quantity of the purified RNA were determined by measuring theabsorbance at 260 and 280 nm using SmartSpec Plus (Bio-Rad). RNA integritywas further verified by 0.015 g mL21 agarose gel electrophoresis.

For each sample, 25 mg of total RNA was used for circRNA-seq librarypreparation. Total RNAwas depleted of ribosomal RNAs using the RiboMinuskit (Illumina). The remaining RNAs were fragmented at 95°C followed by endrepair and 59 adaptor ligation. Then reverse transcription (RT) was performedwith the RT primer harboring a 39 adaptor sequence and randomized hexamer.The cDNAs were purified and subjected to PCR amplification. PCR productscorresponding to 300 to 500 bp were purified and quantified and then stored at280°C until they were used for sequencing.

For high-throughput sequencing, the libraries were prepared following themanufacturer’s instructions (Illumina), and the Illumina NextSeq 500 systemand the HiSeq X Ten system were used for 150-nucleotide paired-end se-quencing by ABlife.

Identification and Quantification of circRNAs

For genome-wide identification of circRNAs, reads containing adapter orpoly-N and low-quality reads were filtered from the raw sequencing reads byin-house Perl scripts. The resultant clean reads were mapped to the rice refer-ence genome (IRGSP v5.0), generating a sequence alignmentmap file. Sequencealignment map files were then used to identify circRNAs with CIRI2 as de-scribed previously (Gao et al., 2015). Based on their genomic origins, circRNAswere classified into three major types: exonic, intronic, and intergenic circR-NAs. For quantification of circRNAs (Song et al., 2016), the numbers of back-spliced reads of each circRNAwas normalized to the total sequencing reads in acorresponding sample data set, defined as reads per million mapped reads.

DEGs and circRNA (DEC) Analysis

The R Bioconductor package edgeR (Robinson et al., 2010) was utilizedto screen out DEGs and DECs. FDR, 0.05 and fold change $ 2 were set as thecut-off criteria for identifying DEGs, and P , 0.05, fold change $ 1.5 foridentifying DECs.

To sort out functional categories of DEGs and genes hostingDECs, GO termsand KEGG pathways were identified using the KOBAS 2.0 server (Xie et al.,2011). A hypergeometric test and the Benjamini-Hochberg FDR control proce-dure were used to define the enrichment of each term.

Analysis of Alternative Splicing in circRNAs

Analysis of alternative splicing was performed using CIRI-AS as previouslydescribed (Gao et al., 2016). CIRI-AS is a novel algorithm for detecting internalcomponents of circRNAs based on split alignment of back-splice junction readpairs and distribution of sequencing depth (Gao et al., 2016).

Genomic DNA, Total RNA Extraction, and RNaseR Treatment

Genomic DNA was extracted from fresh tissue using the cetyl-trimethylammonium bromide method (Murray and Thompson, 1980). Ge-nomic DNA was used as a negative control for PCR with divergent primers.Total RNA was isolated from all samples using TRIzol reagent (Ambion)according to themanufacturer’s protocol. Total RNA sampleswere treatedwithDNase I (NEB) and purified by RNA Clean & Concentrator-25 spin columns(ZYMO Research) to remove DNA contamination and salts. RNA was evaluatedusing 0.02 g mL21 Tris-acetate-EDTA-agarose gel electrophoresis. For RNase-Rtreatment, the purified DNase I-treated total RNA was incubated for 15 min at37°C with 3 units/mg of RNase R (Epicentre) and subsequently purified byphenol-chloroform extraction and reprecipitated in three volumes of ethanol.

PCR Amplification and Sanger Sequencing

Thedivergent and convergentprimerswere designed for circRNAvalidation(Supplemental Table S5). Convergent primerswere used as positive controls forlinear transcripts, and divergent primers were used to detect the candidatecircular template. For each PCR amplification, 20 ng of cDNA or genomic DNAwas used with 23 Phanta Master Mix (Vazyme Biotech). PCR products of theexpected length were dissected from a gel and purified using the GeneJET GelExtraction kit (Invitrogen). Purified PCR products were Sanger sequenced atSangon Biotech or TsingKe Biological Technology Company.

RT-qPCR

For RT-qPCR, first-strand cDNAwas retro-transcribed from total RNAwithorwithout RNase R treatmentwith random sixmers and SuperScript III reversetranscriptase (Invitrogen) and subjected to PCR reactions with the SYBR GreenPCR MasterMix (Takara) on a Bio-Rad CFX Connect Real-Time PCR DetectionSystem. The expression level of circRNAwas normalized to endogenous linearrice ubiquitin (OsUbi, AK059011) transcripts. Each set of experiments was re-peated three times. The primers used for RT-qPCR are listed in SupplementalTable S5.

Plasmid Construction and Overexpression of circR5g05160

The genomic region of circR5g05160 containing the endogenous flankingintrons (Supplemental Fig. S8E) was amplified from LTH genomic DNA withprimers Chr5:2512798j2514806-KpnI-F/SalI-R (Supplemental Table S5) andcloned into a pCAMBIA1300 vector under the control of Cauliflower mosaic virus35S promoter leading to a recombinant plasmid p35S-circR5g05160. For tran-sient expression, Agrobacterium tumefaciens GV3101 cells containing p35S-circR5g05160 were adjusted to optical density at 600 nm 5 0.1 to 0.3 with10 mM MgCl2 and infiltrated into the fully expanded leaves of Nicotiana ben-thamiana. At 2 d postinfiltration, leaves were collected for total RNA extractionand qPCR. Leaves infiltrated with10 mM MgCl2 were used as the control (CK).The production and expression levels of circR5g05160 were examined withdivergent primers of Chr5:2512798j2514806 (Supplemental Table S5). Fulllength of circR5g05160 was validated by Sanger sequencing.

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A. tumefaciens strain EHA105 containing plasmid p35S-circR5g05160was subjected to genetic transformation in rice NPB. Positive transgenic lineswere confirmed by a hygromycin sensitivity test as described previously(Li et al., 2014). Transgenic plants overexpressing circR5g05160 (circR5-g05160OX) were further confirmed by qPCR with the divergent primers ofChr5:2512798j2514806. RT-qPCR was also performed to examine the accumu-lation of linear transcripts from the parent gene LOC_Os05g05160 with theconvergent primers located out of the circR5g05160 region. Primer sequencesare listed in Supplemental Table S5.

Blast Disease Assay

Fully expanded leaves of 3-week-old seedlings were challenged with spores(2 3 105 spore mL21) of M. oryzae eGFP-tagged Zhong8-10-14 (GZ8) throughpunch inoculation or spraying inoculation methods (Park et al., 2012; Li et al.,2019). The disease phenotypewas photographed at 7 d postinoculation. Diseaselesions were measured with ImageJ software. Relative fungal biomass wasquantified as previously described (Park et al., 2012; Li et al., 2019).

Accession Numbers

The raw sequencing data were submitted to NCBI’s Gene Expression Om-nibus and are accessible through GEO series accession number GSE131641.

Supplemental Data

The following supplemental materials are available.

Supplemental Figure S1. Analysis of RNA-seq data from leaves of IR25and LTH.

Supplemental Figure S2. GO enrichment analysis of DEGs between IR25and LTH.

Supplemental Figure S3. Successfully amplified and sequenced circRNAs.

Supplemental Figure S4. KEGG analysis on parent genes of circRNAsidentified in IR25 and LTH.

Supplemental Figure S5. The distribution of the number of splice sites indifferent junctions of circRNAs identified in IR25 and LTH.

Supplemental Figure S6. Expression levels of circRNAs in IR25 and LTH.

Supplemental Figure S7. RT-qPCR validation of differentially expressedcircRNAs in IR25 and LTH.

Supplemental Figure S8. Full-length validation and overexpression strat-egy for circR5g05160.

Supplemental Table S1. Summary of circRNA-seq data from leaves ofblast-resistant and -susceptible rice accessions with or without M. oryzaeinfection.

Supplemental Table S2. High-confidence circRNAs identified in allsamples.

Supplemental Table S3. The 636 circRNAs specifically triggered by M.oryzae infection in IR25 and LTH.

Supplemental Table S4. KEGG enrichment analysis of parent genes pro-ducing circRNAs in IR25 and LTH only upon M. oryzae infection.

Supplemental Table S5. Primers used in this study.

Supplemental Table S6. circRNAs detected in at least two samples and thecorresponding parent genes indicated in Figure 2F.

Supplemental Table S7. PCU of alternative 59 and 39 back-splicing sites.

Supplemental Table S8. Differentially expressed circRNAs in IR25 andLTH upon M. oryzae infection.

ACKNOWLEDGMENTS

We thank Dr. Cai-Lin Lei (Institute of Crop Science, Chinese Academy ofAgricultural Sciences) for providing the seeds of Pikm-monogenic line IR25 and

Dr. Li-Huang Zhu (Institute of Genetics and Developmental Biology, ChineseAcademy of Sciences) for providing the M. oryzae strain Zhong8-10-14.

Received June 13, 2019; accepted October 7, 2019; published October 18, 2019.

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