Supprgssion Of Rieg MiR168 Improvgs Yiglf, Flowgring Timg .
uppression of rice miR168 improves yield,flowering time and immunityHe Wang1,2,5, Yan Li 1,2,5, Mawsheng Chern3,5, Yong Zhu1,2,5, Ling-Li Zhang2, Jun-Hua Lu2, Xu-Pu Li2,Wen-Qiang Dang2, Xiao-Chun Ma2, Zhi-Rui Yang4, Sheng-Ze Yao4, Zhi-Xue Zhao1,2, Jing Fan1,2,Yan-Yan Huang1,2, Ji-Wei Zhang1,2, Mei Pu1,2, Jing Wang1,2, Min He1,2, Wei-Tao Li1,2, Xue-Wei Chen1,2,Xian-Jun Wu1,2, Shi-Gui Li1,2, Ping Li1,2, Yi Li4, Pamela C. Ronald 3 and Wen-Ming Wang 1,2 MicroRNA168 (miR168) is a key miRNA that targetsArgonaute1 (AGO1), a major component of the RNA-inducedsilencing complex1,2. Previously, we reported that miR168expression was responsive to infection by Magnaporthe oryzae, the causal agent of rice blast disease3. However, howmiR168 regulates immunity to rice blast and whether itaffects rice development remains unclear. Here, we report ourdiscovery that the suppression of miR168 by a target mimic(MIM168) not only improves grain yield and shortens flowering time in rice but also enhances immunity to M. oryzae.These results were validated through repeated tests in ricefields in the absence and presence of rice blast pressure. Wefound that the miR168–AGO1 module regulates miR535 toimprove yield by increasing panicle number, miR164 to reduceflowering time, and miR1320 and miR164 to enhance immunity. Our discovery demonstrates that changes in a singlemiRNA enhance the expression of multiple agronomicallyimportant traits.Rice is a staple food for half of the world’s population. Yield,flowering time and disease resistance are key factors in rice production. However, the presence of disease-resistance genes can penalizecrop yield4. For example, a trade-off between biomass and resistancehas been documented in 56% of disease-resistance studies5. There isalso a correlation between growth duration and yield. Crops withhigher yields usually have longer vegetative growth6,7. Despite thesechallenges, recent reports indicate that certain immune regulatorscan promote immunity without yield penalties; in particular, idealplant architecture 1 (ipa1) -1D promotes both yield and immunity8,9.In addition, the presence of a long noncoding RNA has been demonstrated to reduce flowering time without yield penalty10. To date,no regulators have been reported to promote yield, early maturityand immunity together.MicroRNAs (miRNAs) are global gene regulators controllingplant growth, development and immunity11,12. We therefore examined miRNAs that have the potential to affect rice growth, yield andimmunity. miR168 is responsive to Magnaporthe oryzae infection3and targets Argonaute1 (AGO1), which encodes the key component of the RNA-induced silencing complex1,13. For these reasons,miR168 serves as a good candidate for such a study. Here, we created miR168 target mimic (MIM168) transgenic lines and overexpression (OX168) lines (Supplementary Fig. 1a,b). OX168 plantsshowed significantly higher miR168 expression, leading to lowerAGO1 expression, whereas MIM168 displayed significantly lowermiR168 expression, resulting in higher expression of AGO1 compared with the Nipponbare (NPB) control plants (SupplementaryFig. 1b–d).We observed pleiotropic phenotypes in OX168 and MIM168plants. Compared with the NPB control, OX168 plants displayedincreased height, significantly fewer panicles, slightly higher1,000-grain weight and similar grain number per panicle (Fig.1a–c and Supplementary Table 1). Conversely, MIM168 plants wereshorter and displayed significantly more panicles, with similar grainnumber per panicle but slightly lower 1,000-grain weight than thecontrol (Fig. 1a–c and Supplementary Table 1). In addition, OX168lines exhibited an approximately ten-day delay in flowering time anddeveloped 17 leaves on average, whereas MIM168 flowered approximately three days earlier and developed 14 leaves, with the controldeveloping 15 leaves (Fig. 1d,e and Supplementary Fig. 2a,b). Theseresults clearly show that the alteration of miR168 amounts affectsplant architecture and flowering time and may influence yield.To test the capacity of MIM168 and OX168 lines in grain yield,we grew them along with the NPB control in rice fields from 2017to 2019. Three independent lines were included for each of MIM168and OX168. MIM168 lines yielded significantly more grain (up to30–40% higher), calculated both per plant and per area (m2) in therice paddy fields; in contrast, OX168 lines yielded significantly lessgrain (approximately 20–40% lower) (Fig. 1f and SupplementaryTable 1). These results demonstrate that the suppression of miR168changes plant architecture, resulting in higher grain yields.AGO1, the target of miR168, was previously shown to be requiredfor pathogen-associated molecular-pattern-triggered immunity inArabidopsis14. We therefore tested two lines for each of MIM168 andOX168 in a rice field with high rice blast pressure to assess theirpotential effects on resistance to M. oryzae. We found that, whilethe NPB control yield was 13% less under blast disease pressure,MIM168 lines had only a 1–4% reduction in yield. In contrast,OX168 lines displayed a 20–40% reduction in yield (Fig. 2a,b andSupplementary Fig. 2c). These results suggest that lower miR168levels enhance resistance, whereas higher miR168 levels decreaseresistance to M. oryzae. Under blast nursery conditions, MIM168lines yielded up to 75% higher per m2 than the NPB control (Fig. 2a,band Supplementary Fig. 2c). Consistent with the field results, OX168State Key Laboratory of Crop Gene Exploration and Utilization in Southwest China, Sichuan Agricultural University, Chengdu, China. 2Rice ResearchInstitute and Key Lab for Major Crop Diseases, Sichuan Agricultural University, Chengdu, China. 3Department of Plant Pathology, University of CaliforniaDavis, and the Joint BioEnergy Institute, Davis, CA, USA. 4The State Key Laboratory of Protein and Plant Gene Research, College of Life Sciences, PekingUniversity, Beijing, China. 5These authors contributed equally: He Wang, Yan Li, Mawsheng Chern, Yong Zhu. e-mail: j316wenmingwang@sicau.edu.cn1Nature Plants VOL 7 February 2021 129–136 www.nature.com/natureplants129
LettersNATuRe PlAnTsb-1-2-3NPBMIM168c-2-1P 0.0001P 0.00015P 0.0001100-3NPMIM BM 168IM -11M 68IM 216O 8-3X16O 8-1X16O 8-2X168-3NPB15P 0.0004Panicle numberper plant20P 0.000125P 0.001aOX168e30P 0.01016O 8-3X16O 8-1X16O M168-3OX168-3P 0.004P 0.0001P 0.004P 0.0001P 0.0001P 0.0001P 0.002P 0.046P 0.002Yield per m2 area inpaddy field (kg)P 0.00011.0MIM168-1P 0.0001P 0.002P 0.001NPBIMNPBM 168IM -11M 68IM 216O 8-3X16O 8-1X16O 8-2X168-370M-3P 0.0001P 0.002P 0.0001P 0.0001P 0.000180-2MIM1681.5P 0.000190P 0.0001100-1IMMIM168-NPBf110P 0.041Days to floweringdMMIM16NPB10P 0.0001P 0.002P 0.00015P 0.02710P 0.017P 0.00115P 0.000120P 0.00011,000 grain weight250201720182019YearFig. 1 miR168 regulates rice yield and flowering time. a, Gross morphology and husked grains of the NPB control, OX168 and MIM168. Scale bars, 50 cmfor gross morphology and 5 mm for grains. b–d, Quantification of panicle number (b), 1,000 grain weight (c) and flowering time (d). The data are shown asmean s.d. (n 5 independent plants). e, Panicle morphology. Scale bar, 5 cm. f, Yields of the NPB control, OX168 and MIM168 per 1 m2 area in the paddyfields in 2017, 2018 and 2019. Each dataset contains three plots. The data are shown as mean s.d. (n 3 biologically independent plots). For b,c,e, andf, the P values were determined by one-way analysis of variance (ANOVA).lines were more susceptible to M. oryzae strain Guy11, as well as97-27-2 and NC-34 (two strains isolated from rice fields in northChina and south China, respectively). The OX168 lines displayedsignificantly larger disease lesions and 2–4.5-fold higher fungalbiomass, whereas MIM168 plants were more resistant, displayingsmaller lesions and less than 50% fungal biomass compared withthe NPB control (Fig. 2c,d and Supplementary Fig. 2d,e). Moreover,OX168 showed significant increased invasive hyphae progression at18–48 hours post-inoculation (hpi) with Guy11 in sheath cells andreduced H2O2 accumulation at 48 hpi. In contrast, MIM168 showeddelayed invasive hyphae progression and increased H2O2 accumulation at the infected sites (Fig. 2e,f), a marker of defence responses.Consistently, MIM168 lines accumulated higher mRNA levels ofthe defence-related genes, including OsPR1, OsPR10b, Os04g10010and the H2O2 production genes respiratory burst oxidase homologues130RbohB and RbohE, but lower levels of a catalase gene encoding H2O2degradation enzyme (Supplementary Fig. 2f–k). OX168 lines, however, displayed an opposite or unchanged expression pattern of thesegenes in response to M. oryzae infection. These results demonstratethat miR168 suppresses rice immunity against M. oryzae, and thesuppression of miR168 enhances rice immunity to M. oryzae.We next created AGO1-silencing transgenic lines (AGO1i) toexplore whether miR168 regulates rice growth and immunity viaAGO1. AGO1i lines showed significantly lower AGO1 (AGO1a-d)amounts compared with the NPB control (Supplementary Fig. 3a,b).Consistent with the yield traits of OX168, AGO1i lines showedtaller plants, slightly larger seeds, fewer tillers, lower 1,000-grainweights and significantly reduced yields than the NPB control(Supplementary Fig. 3c–e and Supplementary Table 1). Moreover, asobserved for the OX168 lines, the AGO1i lines displayed enhancedNature Plants VOL 7 February 2021 129–136 www.nature.com/natureplants
LettersNATuRe PlAnTsbaNormal paddy yardBlast nursery–13.0%0.8% 0.26%4.0% 1.22%13.3% 1.22%5.9% 1.58%45.4% OX168-2MIM168-24P 0.0001P 0.0001P 0.0001P 0.041P 0.012P 0.028Relative fungal biomass6P 0.0001P 0.028P 0.001P 0.004OX168NPBP 0.0001P 0.0001cBlast nurseryP 0.0001P 0.0001P 0.0001P 0.074P 0.026P 0.007P 0.0001P 0.0001P 0.0001P 0.0001–20.6%NPBMIM1681MIM1682OX168-1OX168-201.4% 0.31%–39.4%1.00.5P 0.019P 0.00011.5P 0.0001P 0.0001Normal paddy yard–1.4% –4.2%P 0.723Yield per m2 (kg)2.0NPBOX168-197-27-2NC-34MIM168-112 hpifAppressorium did not infect first cellInfected first cellExtended to second cellExtended to third and more cells24 hpi36 hpi48 IM168-1243648hpi020406080Percentage of infection phases (%)100Fig. 2 miR168 regulates rice immunity against M. oryzae. a, Yields of the NPB control, OX168 and MIM168 with or without blast disease pressure (blastnursery) in 2019. The bars indicate mean s.d. (n 3 biologically independent plots). b, Blast disease severities of the NPB control, OX168 and MIM168 infields with or without blast disease pressure in Wenjiang, Sichuan Province, China. The percentages indicate M. oryzae-infected panicles to total countedpanicles. c, Disease lesions of detached leaves at five days post-inoculation (dpi) by punch inoculation with three blast strains. Scale bars, 5 mm. d, qPCRof fungal DNA of the samples in c. The data are shown as mean s.d. (n 3 independent samples). e, Microscopic images showing the infection status ofGFP-tagged Guy11 at 12, 24, 36 and 48 hpi. Scale bars, 20 μm. The white arrows indicate appressoria, and the red arrows indicate invasive hypha. H2O2 incells was stained by DAB, and the brownness intensity indicates the H2O2 amount in the cells. f, Quantification of pathogenesis of Guy11. Over 150 conidiain each line were analysed in three independent experiments. For a and d, the P values were determined by one-way ANOVA.susceptibility to and accelerated invasive progression of M. oryzae,and less H2O2 accumulation upon inoculation with three strains(Supplementary Fig. 3f–i). These results indicate that AGO1 mediates miR168 function.Because the miR168–AGO1 module regulates the accumulation of miRNAs globally, we performed small RNA sequencing toexamine changes of miRNAs in leaves at the seedling and tilleringstages and in panicles at the booting stage. Many miRNAs alteredexpression in OX168 and MIM168 lines (Supplementary Table 2).Nature Plants VOL 7 February 2021 129–136 www.nature.com/natureplantsWe found 162 miRNAs at the seedling stage, 156 at the tilleringstage and 197 at the booting stage that were regulated by miR168(Supplementary Fig. 4a and Supplementary Table 3). Among them,we found 15 miRNAs11,15–17 that were previously identified as regulators of rice developmental processes (Supplementary Fig. 4b).To identify the miR168–AGO1 module-regulated miRNAs thatare probably involved in immunity, we compared those miRNAsaffected in MIM168 and OX168 (Supplementary Table 3) withthose responsive to M. oryzae (Supplementary Table 2 in ref. 3) and131
LettersNATuRe PlAnTsRelative miR1320 levelP 0.001P 0.001P 0.003P 0.001O KXO 3 aM X320IM 2 1M 13 0-2IM 213 020 1-2P 0.001P 0.001P 0.919P 0.664P 0.914P 0.001P 0.999P 0.0001P 0.00010.40GZ897-27-2P 0.0001P 0.0001P 0.992P 0.0001P 0.007Relative fungal )OX1320/OX168-1OX1320/OX168-2P 0.9999h-2P 0.0001P 0.0001-1P 0.994OX1320/MIM168(–)P 0.0001NC-346420P 0.00397-27-220P 0.999Guy1140P 0.0001P 0.0001MIM1680P 0.968-20.560P 0.0001P 0.0001-1P 0.0001P 0.0001P 0.0001P 0.0001NC-34OX1320/OX168P 0.0001P 0.0001P 0.0001P 0.00011P 0.0001P 0.0001P 0.0001P 0.0001Relative fungal elative miR1320 levelKaGZ8(–)1.0f0OX1681.512 24 48Time (hpi)e-23g200X1O 32X 0 OO 13 /OX X1X1 20 1 632 /O 68 80/ X1 (–OO 6 )X1X1 86 1O 32X1 0/ M 8-2O 3 MI IMX1 20 M 132 /M 16 680/ IM 8(–M 1 )IM 6816 -182-10300OMIM1320-212 24 48Time (hpi)P 0.997OX1320-1Guy11Ka00P 0.0001P 0.0001d0Tillering stage2P 0.999Seedling stage4P 0.0001P 0.000100.56P 0.96811.0cIRBLkm-TsMockM. oryzae8P 0.364P 0.0001P 0.0012P 0.018P 0.040P 0.0001P 0.0001P 0.004P 0.034P 0.000131.5P 0.0001P 0.00014OX168-2MIM168-2LTHMockM. oryzaeP 0.9995Relative miR1320 levelbNPBOX168-1MIM168-1Relative miR1320 levelaGuy11Fig. 3 miR1320 contributes to miR168-regulated immunity. a, RT–qPCR of miR1320 in the NPB control, OX168 and MIM168. b, RT–qPCR of miR1320 ina susceptible variety (LTH) and a resistant variety (IRBLkm-Ts) upon M. oryzae infection or mock treatment. The data were normalized so that the levelat 0 hpi was arbitrarily set as 1. c, RT–qPCR of miR1320 in the Kasalath (Ka) control, OX1320 and MIM1320. d, Blast lesions of detached leaves at 5 dpiwith three blast strains. Scale bars, 5 mm. e, qPCR of fungal DNA of the samples in d. Kasalath is used as the control. f, RT–qPCR of miR1320 in OX168,OX1320/OX168, OX1320/OX168 null segregant ( ), MIM168, OX1320/MIM168 and OX1320/MIM168 null segregant ( ). OX168 and MIM168 are usedas controls, respectively. g, Blast lesions of detached leaves at 5 dpi with three blast strains. Scale bars, 5 mm. h, qPCR of fungal DNA of the samples ing. For the P value analysis, the data were compared with those of OX168 and MIM168, respectively. For a–c,e,f and h, the data are shown as mean s.d.(n 3 independent samples). All the P values were determined by one-way ANOVA.identified 54 miRNAs from 39 families. Among these, we foundnine miRNAs3,12,18–23 that were previously identified to be involvedin immunity (Supplementary Fig. 4b,c and Supplementary Table 4).Consistently, the transcript levels of many target genes of these ninemiRNAs were changed in OX168 and MIM168 (SupplementaryFig. 4d). These data indicate that many miRNAs mediate miR168function, and some of these are critical to immunity.First, the previously uncharacterized miR1320 was upregulated inMIM168 and downregulated in OX168 at the seedling and tilleringstages. Its expression was validated by quantitative PCR with reversetranscription (RT–qPCR) and northern blotting analysis, althoughnorthern blot seems not sensitive enough to detect the alteration of low-abundance miR1320 (Fig. 3a, Supplementary Fig. 4eand Supplementary Table 3). Importantly, miR1320 was differentially upregulated in a blast-resistant rice variety (International132Rice Blast Line Pyricularia-Kanto51-m-Tsuyuake (IRBLkm-Ts)),consistent with a previous report24, whereas it was downregulatedin the susceptible variety Lijiang xin Tuan Heigu (LTH) upon M.oryzae infection (Fig. 3b). These results suggest that miR1320 maymediate miR168 function in immunity to M. oryzae. We thereforetested the effects of miR1320 directly by generating overexpression(OX1320) and target mimic (MIM1320) lines (Fig. 3c) in Kasalath,and overexpression lines in OX168 (OX1320/OX168) and MIM168(OX1320/MIM168) (Fig. 3f). We found that OX1320 lines showedenhanced resistance, with smaller lesions and lower fungal biomass(by 50%), whereas MIM1320 displayed the opposite phenotypes(Fig. 3d,e and Supplementary Fig. 5a,b). Moreover, OX1320/OX168lines were more resistant than OX168 and null segregants, andOX1320/MIM168 lines were more resistant than MIM168 and nullsegregants (Fig. 3g,h). Consistently, OX1320 lines showed delayedNature Plants VOL 7 February 2021 129–136 www.nature.com/natureplants
LettersNATuRe PlAnTsP 0.0001P 0.0002P 0.001P 0.0002NPB -1SPL1410NAC11miR1642NAC11miR164105Hd3aRFT1P 0.00215OiNPBP 0.043P 0.999P 0.015P 0.00402003OX164-2MIM164-2P 0.9999P 0.439P 0.0001P 0.0011220P 0.0001P 0.0001P 0.0001P 0.00012OX164-1MIM164-1P 0.0001P 0.0001P 0.0001P 0.00013NPB240P 0.600P 0.301P 0.0001P 0.00014P 0.056P 0.982P 0.007P 0.001Relative levelhNPBOX168-1OX168-2MIM168-1MIM168-25Relative levelg200O NPXO 53 BM X53 5-1IM M5 5-2IM 3553 -1520OX535/MIM168MIM168 (–)-1-2X5O 35 OXO 53 /OX X1X5 5 1 635/OX 68( 8/O 16 –)OX1 8X5O 35 M 68-1X5 /M I 2O 3 IM MX5 5 1 135/MI 68 68/M M1 (–IM 68 )16 -18210P 0.995P 0.000120P 0.000130P 0.999OX168-2P 0.0001fOX535/OX168(–)-1-2Panicle numberper plant40-2 -1OX535 MIM535P 0.995miR535P 0.0001Relative level0Late tillering stageePanicle numberper plantd2P 0.0001Early tillering stage9031SPL14miR53595P 0.995SPL14miR535100MIM535-1MIM535-2P 0.0001P 0.0001P 0.0001P 0.00010P 0.0001P 0.00010.5cNPBOX535-1OX535-2105P 0.0001P 0.0001P 0.0001P 0.00011.0P 0.0001P 0.0001Relative level inshoot meristem1.5bOX168-2MIM168-2OX168-1MIM168-1P 0.0001P 0.0001P 0.0001P 0.0001NPB2.0P 0.0001P 0.0001P 0.02P 0.0003aOX164-1 -2NPB-1MIM164-2Booting stagejklOX164/MIM168-1P 0.0001OX164/MIM168-2P 0.0001P 0.000170nac11-2P 0.000180nmP 4/MIM168-1-2Days to floweringZH11nac11-1MIM168MIM168O NPXO 16 BX 4M 16 -1I 4M M16 -2IM 416 -14270P 0.000180P 0.000190P 0.0001Days to iR164GrowthdurationDays to floweringFig. 4 miR535 and miR164 contribute to miR168-regulated development. a, RT–qPCR of miR535 and SPL14 mRNA in the shoot meristem of the NPBcontrol, OX168 and MIM168. b, RT–qPCR of miR535 and SPL14 mRNA in the NPB control, OX535 and MIM535. c, Gross morphology (top; scale bar,50 cm), husked grains (middle; scale bar, 5 mm) and panicle morphology (bottom; scale bar, 5 cm) of the indicated lines. d, Panicle numbers of theNPB control, OX535 and MIM535. e, Gross morphology of OX168, OX535/OX168, OX535/OX168 null segregant ( ), MIM168, OX535/MIM168 andOX535/MIM168 null segregant ( ). f, Panicle numbers of the indicated lines. OX168 and MIM168 are used as independent controls. g, RT–qPCR ofmiR164 and NAC11 mRNA in the NPB control, OX168 and MIM168 at the booting stage. h, RT–qPCR of miR164 and mRNAs of NAC11, Hd3a and RFT1in the NPB control, OX164 and MIM164. i,j, Gross morphology (i; scale bars, 50 cm) and flowering time (j) of the NPB c
LETTERS https://.org10.103841477-021-00852-x 1Stat e at w ersity heng 2Ric esear Institut e ersity heng 3Depar athology ersit Da a 4T e at rot esear olleg University 5T ontribut w j316wenmingwang@sicau.edu.cn MRNA168 (R168) e RNA Argonaute1 (AGO1), RNA- omplex 1,2.Previously, eport R168
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