Screening of Brown Planthopper Resistant miRNAs in Rice and Their Roles in Regulation of Brown Planthopper Fecundity

doi:10.1016/j.rsci.2022.05.003

Abstract

Abstract:

MicroRNAs (miRNAs) can participate in plant-insect interactions, which regulate plant defense networks. In this study, we analyzed the miRNA expression profiles of six rice varieties before and after brown planthopper (BPH)-feeding. We identified 45 differentially expressed miRNAs between BPH- susceptible and BPH-resistant rice varieties and 144 miRNAs that responded to BPH-feeding. Thus, miRNAs may be involved in multiple pathways regulating rice defense response against BPH. In addition, we found that the genetic history of rice varieties determined the regulation mode of the miRNA and affected the amounts, types, changing trends and response periods of miRNAs in response to BPH- feeding. To conclude, we scanned seven potential cross-kingdom miRNAs, of which miR5795 may target the vitellogenin gene in BPH, causing a 16.07% reduction in BPH oviposition. The results provide new miRNA information of rice-BPH interactions and BPH-resistant rice variety breeding.

Key words: Nilaparvata lugens, insect-resistant rice, miR5795, fecundity

Lü Jun, Liu Jinhui, Chen Lin, Sun Jiawei, Su Qin, Li Shihui, Yang Jianhua, Zhang Wenqing. Screening of Brown Planthopper Resistant miRNAs in Rice and Their Roles in Regulation of Brown Planthopper Fecundity[J]. Rice Science, 2022, 29(6): 559-568.

Figures/Tables 6

Fig. 1. miRNAs differentially expressed in brown planthopper (BPH)-susceptible and BPH-resistant rice varieties before BPH-feeding. A, Principal component analysis (PCA) of miRNAs in rice samples before BPH-feeding. B, Volcano plot of differential miRNA expression in BPH-resistant and BPH-susceptible rice varieties. ‘down’ and ‘up’ mean miRNA expression was down-regulated and up-regulated in BPH-resistant rice varieties compared with BPH-susceptible rice varieties, respectively, and ‘non’ means the expression of miRNA was not significantly different between the BPH-resistant and BPH-susceptible rice varieties. C, Heatmap of differential miRNA expression profile. D, Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment of target genes of differential miRNAs. RH, Rathu Heenati.

Table 1. Summary of rice miRNAs in response to brown planthopper (BPH)-feeding.

Rice variety	After BPH-feeding at 8 h / Before BPH-feeding (0 h)
TN1	miR5792	miR5489	miR160f-5p	miR3980b-5p
	miR531c	miR1861l
	miR5501	miR5801a	miR5795	miR1882e-3p
Nipponbare	miR160e-5p	miR160f-5p	miR393b-3p	miR160d-5p
Nipponbare	miR394
IR36	miR1850.3	miR2863b	miR6249b	miR5337a
	miR2876-5p	miR2876-3p	miR1846b-3p	miR2865
	miR2092-3p	miR5795	miR418
	miR3980b-5p	miR531c	miR399h	miR398b
	miR1850.1	miR156l-5p	miR3980b-3p	miR1320-3p
Mudgo	miR528-3p	miR168b	miR167h-3p	miR171e-5p
Mudgo	miR2102-3p	miR171d-5p	miR5809
RH	miR1428e-3p	miR1428g-5p	miR319b
	miR5078	miR398b	miR393b-3p	miR1430
	miR166j-5p	miR5077	miR164f	miR169g
	miR396f-3p	miR167j	miR1432-5p	miR169r-3p
	miR156j-3p	miR168a-5p	miR169i-5p.2	miR166d-5p
	miR162b	miR156l-3p	miR399d	miR169r-5p
	miR810b.2	miR396c-5p	miR444f	miR397a
	miR171h	miR393b-5p	miR7693-5p	miR397b
	miR444c.1	miR810a	miR810b.1	miR169e
	miR169c	miR164d	miR159b	miR169q
Rice variety	After BPH-feeding at 32 h / Before BPH-feeding (0 h)
TN1	miR3979-5p	miR398b	miR3980b-5p	miR531c
	miR2876-3p
IR36	miR2863b	miR1320-3p	miR5805	miR2092-3p
	miR6256	miR5806	miR398b	miR5535
	miR2922	miR169q	miR171e-5p	miR3979-3p
R476	miR169e	miR5830	miR6255	miR171a
	miR159d	miR396b-3p	miR5486	miR2877
	miR5490	miR5081	miR2106	miR827
	miR1882h	miR1882e-3p	miR156j-5p	miR438
	miR169a	miR1320-3p	miR5822	miR5527
	miR390-3p	miR6253	miR169p	miR164d
	miR5151	miR417	miR156l-5p	miR5494
	miR5523	miR160d-3p	miR5076	miR5539b
	miR5073	miR171d-5p	miR5075	miR5829
	miR1850.1	miR166k-5p	miR2100-5p	miR166h-5p
	miR169r-3p	miR166e-5p	miR160f-3p	miR1861c
	miR395o	miR167a-3p	miR1850.2	miR166b-5p
	miR2928	miR810a	miR5790	miR171e-5p
	miR2102-3p
Mudgo	miR171a	miR5517	miR1859	miR2106
	miR5511	miR160d-3p	miR166e-3p	miR1320-3p
	miR528-3p	miR396c-3p	miR2930	miR2102-3p
	miR2926	miR171e-5p	miR6246	miR5073
	miR171d-5p	miR408-5p	miR5153	miR168b
	miR167h-3p	miR2924	miR6249b	miR160f-5p
	miR1850.1	miR167i-3p	miR1858b	miR160f-3p
	miR5809	miR1437a	miR166l-5p	miR1846d-5p
RH	miR529b	miR535-5p
	miR396c-3p	miR166k-5p	miR171d-5p	miR530-3p
	miR166h-5p	miR160f-3p	miR172d-5p	miR810a
	miR5073	miR1850.1	miR166d-5p	miR6246
	miR166b-5p	miR5485	miR166e-5p	miR171e-5p

Table 1. Summary of rice miRNAs in response to brown planthopper (BPH)-feeding.

Rice variety	After BPH-feeding at 8 h / Before BPH-feeding (0 h)
TN1	miR5792	miR5489	miR160f-5p	miR3980b-5p
	miR531c	miR1861l
	miR5501	miR5801a	miR5795	miR1882e-3p
Nipponbare	miR160e-5p	miR160f-5p	miR393b-3p	miR160d-5p
Nipponbare	miR394
IR36	miR1850.3	miR2863b	miR6249b	miR5337a
	miR2876-5p	miR2876-3p	miR1846b-3p	miR2865
	miR2092-3p	miR5795	miR418
	miR3980b-5p	miR531c	miR399h	miR398b
	miR1850.1	miR156l-5p	miR3980b-3p	miR1320-3p
Mudgo	miR528-3p	miR168b	miR167h-3p	miR171e-5p
Mudgo	miR2102-3p	miR171d-5p	miR5809
RH	miR1428e-3p	miR1428g-5p	miR319b
	miR5078	miR398b	miR393b-3p	miR1430
	miR166j-5p	miR5077	miR164f	miR169g
	miR396f-3p	miR167j	miR1432-5p	miR169r-3p
	miR156j-3p	miR168a-5p	miR169i-5p.2	miR166d-5p
	miR162b	miR156l-3p	miR399d	miR169r-5p
	miR810b.2	miR396c-5p	miR444f	miR397a
	miR171h	miR393b-5p	miR7693-5p	miR397b
	miR444c.1	miR810a	miR810b.1	miR169e
	miR169c	miR164d	miR159b	miR169q
Rice variety	After BPH-feeding at 32 h / Before BPH-feeding (0 h)
TN1	miR3979-5p	miR398b	miR3980b-5p	miR531c
	miR2876-3p
IR36	miR2863b	miR1320-3p	miR5805	miR2092-3p
	miR6256	miR5806	miR398b	miR5535
	miR2922	miR169q	miR171e-5p	miR3979-3p
R476	miR169e	miR5830	miR6255	miR171a
	miR159d	miR396b-3p	miR5486	miR2877
	miR5490	miR5081	miR2106	miR827
	miR1882h	miR1882e-3p	miR156j-5p	miR438
	miR169a	miR1320-3p	miR5822	miR5527
	miR390-3p	miR6253	miR169p	miR164d
	miR5151	miR417	miR156l-5p	miR5494
	miR5523	miR160d-3p	miR5076	miR5539b
	miR5073	miR171d-5p	miR5075	miR5829
	miR1850.1	miR166k-5p	miR2100-5p	miR166h-5p
	miR169r-3p	miR166e-5p	miR160f-3p	miR1861c
	miR395o	miR167a-3p	miR1850.2	miR166b-5p
	miR2928	miR810a	miR5790	miR171e-5p
	miR2102-3p
Mudgo	miR171a	miR5517	miR1859	miR2106
	miR5511	miR160d-3p	miR166e-3p	miR1320-3p
	miR528-3p	miR396c-3p	miR2930	miR2102-3p
	miR2926	miR171e-5p	miR6246	miR5073
	miR171d-5p	miR408-5p	miR5153	miR168b
	miR167h-3p	miR2924	miR6249b	miR160f-5p
	miR1850.1	miR167i-3p	miR1858b	miR160f-3p
	miR5809	miR1437a	miR166l-5p	miR1846d-5p
RH	miR529b	miR535-5p
	miR396c-3p	miR166k-5p	miR171d-5p	miR530-3p
	miR166h-5p	miR160f-3p	miR172d-5p	miR810a
	miR5073	miR1850.1	miR166d-5p	miR6246
	miR166b-5p	miR5485	miR166e-5p	miR171e-5p

Fig. 2. Different miRNAs participate in rice-brown planthopper (BPH) interactions after BPH-feeding. A, UpSet diagram of miRNAs in rice response to BPH-feeding. ‘●’ indicates that the miRNAs appeared in only one rice variety, and ‘●-●’ indicates that the miRNAs appeared in two or more rice varieties. B, UpSet diagram of miRNA target genes in rice response to BPH- feeding. ‘●’ indicates that the miRNA target genes appeared in only one rice variety, and ‘●-●’ indicates that the miRNA target genes appeared in two or more rice varieties. C, Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment pathway is shared by four BPH-resistant rice varieties. NB, Nipponbare; RH, Rathu Heenati.

Fig. 3. Screening of potential cross-kingdom miRNAs in rice. A, Venn diagram of two sets of miRNAs. B, Venn diagram of target prediction of rice miRNAs using three bioinformatic algorithms (miRanda, TargetScan and RNA22) based on the total transcripts of Nilaparvata lugens. C, Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment of target genes in N. lugens of miRNAs.

Table 2. Number of targets of rice miRNAs on fecundity of Nilaparvata lugens.

miRNA name	No. of sequences in coding sequence region			No. of sequences in 3′-untranslated region
miRNA name	miRanda	RNA22	TargetScan	miRanda	RNA22	TargetScan
miR1428g-5p	2	1	2	2	1	3
miR1432-5p	2	2	3	0	1	1
miR168b	4	12	3	1	2	0
miR1882h	4	4	2	2	0	0
miR2876-5p	7	8	5	0	0	1
miR3980b-5p	7	0	4	0	0	1
miR5795	8	11	8	0	1	0

Fig. 4. miR5795 in rice may target vitellogenin gene in brown planthopper (BPH) and subsequently decrease its fecundity. A-C, The number of eggs per pair (A), hatching rate (B) and the number of hatched eggs per pair (C) of Nilaparvata lugens after injection of miR5795 mimics. D, Predicted binding sites between miR5795 and NlVg with energy of -87.65 kJ/mol. E, Dual-luciferase assay between miR5795 and NlVg. psiCHECK-2, Vector without insertion sites; WT, Wild type vector; MT, Mutation type vector. F, mRNA expression level of NlVg in whole bodies of BPH after injection of miR5795 mimics at 24, 48 and 72 h. G, Western blot for NlVg in whole bodies of BPH after injection of miR5795 mimics at 24, 48 and 72 h. β-actin was used as an internal reference. H, Protein expression levels of NlVg in whole bodies, ovaries and fat bodies of BPH after injection of miR5795 at 72 h. Data are Mean ± SE. n = 3 in A-C, F and H, and n = 9 in E. *, P < 0.05; **, P < 0.01.

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