mRNA cleavage by 21-nt phasiRNAs dependent on AGO1d is important for temperature-sensitive male sterility in rice
Temperature-sensitive male sterility is one of the core components for hybrid rice breeding based on two-line system. We previously found that knockout of ARGONAUTE 1d (AGO1d) caused temperature-sensitive male sterility in rice by influencing phased small interfering RNA (phasiRNA) biogenesis and function. However, the specific phasiRNAs and their targets underlying the temperature-sensitive male sterility in ago1d mutant remain to be determined. Here, we demonstrate that the ago1d mutant displays normal female fertility but complete male sterility at low temperature. Through a multi-omics analysis of small RNA, degradome, and transcriptome, we found that 21-nt phasiRNAs account for the greatest proportion of the 21-nt small RNA (sRNA) species in rice anthers and are sensitive to low temperature and markedly down-regulated in ago1d mutant. Moreover, we found that 21-nt phasiRNAs are essential for the mRNA cleavage of a set of fertility- and cold tolerance-associated genes, such as EDT1, TDR, OsPCF5 and OsTCP21, directly or indirectly determined by AGO1d-mediated gene silencing. The loss-of-function of 21-nt phasiRNAs can result in up-regulation of their targets and causes varying degrees of defects in male fertility and grain setting. Our results highlight the essential functions of 21-nt phasiRNAs in temperature-sensitive male sterility in rice and suggest their promising application in two-line hybrid rice breeding in the future. Overall design: Anthers at S8-9 of wild type and ago1d mutant grown at 28 °C and 22 °C were collected with three biological replicates. The degradome libraries were constructed as previously described (German et al., 2009) with minor modifications. Total RNA was isolated from each sample and purified with oligo(dT) beads. The purified poly(A) RNAs containing a 5' monophosphate of the 3' cleavage product of the mRNA were ligated to 5'-adaptor. First-strand cDNA was reversely transcribed using 3'-adapter random primers, and size selection was performed with AMPureXP beads. Then the cDNAs were amplified with PCR by the following conditions: initial denaturation at 95 °C for 3 min; 15 cycles of denaturation at 98 °C for 15 sec, annealing at 60 °C for 15 sec, and extension at 72 °C for 30 sec; and then final extension at 72 °C. The final products were sequenced (single-end 50-bp) on an Illumina Hiseq2500 platform by LC-BIO (Hangzhou, China). Degradome sequencing data were preprocessed by removing adapters and then mapped to the Oryza sativa v.7.0 genome sequences (MSU Rice Genome Annotation Project) using bowtie. Target prediction and validation of the 3,116 sRNAs with the degradome sequencing was conducted by CleaveLand4 and GSTAr which are available at https://sites.psu.edu/axtell/software/cleaveland4/. The validated targets were defined with mismatched score = 7 (specifically, mismatched query bases or target-bulged bases, are penalized 1. G-U wobbles are penalized 0.5. These penalties are double within positions 2-13 of the query.), category = 2 (category 0: >1 read, equal to the maximum on the transcript, when there is just 1 position at the the maximum value; category 1: >1 read, equal to the maximum on the transcript, when there is >1 position at maximum value; category 2: >1 read, above the average depth, but not the maximum on the transcript; category 3: >1 read, but below or equal to the average depth of coverage on the transcript; category 4: Just one read at that position) and read counts >1 in wild type at both 28 °C and 22 °C.