Peer review of: Dicer-like 5 deficiency confers temperature-sensitive male sterility in maize (2018)

Reviewers' comments: 

Reviewer #1 (Remarks to the Author): 

The exact functions of DCL5 and 24nt-phasiRNAs in reproductive organs of monocots have been a 
mystery since their discoveries. In this paper, the authors confirmed the involvement of DCL5 in 
24-nt phasiRNA biogenesis and showed that DCL5 and 24-nt phasiRNAs play vital roles in anther 
developments only at high temperatures. The thermo-sensitivity and the tapetal cell defects of 
dcl5-1 mutant are well described. In general, the manuscript is well written and the amount of 
work presented in the paper is appreciated. 

A few major comments are listed below. 
1. It is surprising to see that monocot plants make such efforts to cope with a possible condition of 
higher temperatures during reproduction. Why is this mechanism not evolved in dicot? 

2. If DCL5 and 24-nt phasiRNAs are indispensable under higher temperature, is DCL5 expression 
or 24-nt phasiRNA production influenced by elevated temperature? 

3. Given the fact that normal tetrad cells were observed in the dcl5-1 mutant (Fig S4) but inviable 
pollens were seen later (Fig S2), it is interesting to determine what stage of gametogenesis is 
affected? 

4. Related to the last point, the earliest phenotype reported here is the abnormal tapetal cells in 
1.5 mm anther. Although the meiosis seems to be normal, suggesting that abnormality of tapetal 
cells may not affect meiosis, I would be very conservative to exclude the possibility that DCL5 and 
24nt-sRNAs having function in meiosis. 

5. It will be interesting to know that whether programmed cell death of tapetal cells is affected in 
dcl5 mutant. 

6. All tapetal cells would become binucleate at end of meiosis. Do slightly larger anthers (2.5-
3.5mm) in dcl5-1 mutant also have fewer binucleate tapetal cells? This data would clarify whether 
tapetal development delayed or arrested. 

7. Line 76-78. “we conclude that the 24-nt phasiRNA pathway is localized in tapetal cells” might be 
over-stated if the precursor of 24-nt phasiRNA are considered to be part of the pathway. Since 
according to Zhai et al. (2015) 24-PHAS are present in tapetal cells and in meiocytes. 

Minor points 
1. Line 16: miRNA needs an abbreviation. 
2. Fig S1 does not contain mRNA abundance data of dcl5 alleles. 
3. Fig S4, dcl5-1 mutant meiocytes have more organelle? what are those tiny DAPI-stained dots? 
4. Line 76, (4)? 
5. Fig S7, dcl5-1 mutant under permissive and restrictive temperature showed higher expression 
of non-phasi 24-nt sRNAs.?? What they are? Why it is not seen in the dcl5-1 mutant at the 1.5 or 
2 mm anthers? 
6. Line 40-47: I suppose that dcl5-2, dcl5-3 and dcl5-4 plants are sterile, based on descriptions in 
lines 84-86. The authors should mention whether these three alleles are sterile or fertile with data 
shown in supplemental figures. 
7. Figure A-E, G-K in Figure S4 should be enlarged for easier reading. 
8. Both dcl5-2 and dcl5-3 allele have normal DCL5 expression but without 24nt phasiRNAs (Figure 
S1). It would be an interesting to examine whether any phenotypic difference can be observed in 
tapetal cells and meiosis. 
9. Fig S5 is incomplete. 

Reviewer #2 (Remarks to the Author): 

The manuscript, entitled “Dicer-like 5 deficiency confers temperature-sensitive male sterility in 
maize” (Zhang, et al) reports novel findings regarding the role that DICER-LIKE 5 plays in 
maintaining maize male fertility. DICER-LIKE (DCL) proteins are essential for biogenesis of small 
regulatory RNAs in angiosperms. Unlike other members (DCL1, DCL2, DCL3, and DCL4) that are 
conserved across all flowering plants, DCL5 was recently found monocot-specific. In this 
manuscript, authors created maize Dcl5 mutations using CRISPR-Cas9 gene editing technique, 
characterized maize dcl5 mutants under different temperature-controlled growth condition and 
discovered that maize DCL5 is essential for robust male fertility. On the molecular level, this paper 
suggests that DCL5 corresponds to biogenesis of 24-nt phasiRNAs, and the phasiRNA pathway is 
believed localized in tapetal cells and plays important roles in plant reproductive process. Findings 
from this study has not only advanced our understanding of DCL5’s role in maintaining a robust 
male fertility and responding to the temperature changes, but also has significance in agricultural 
practice. I only have some minor suggestion and comments. 

1. By looking at the Figure 2, which seems to me that dcl5-1 mutant anthers are not only 
developing less binucleate tapetal cells, but also abnormal in cell number and size. I am not sure if 
this is just from the particular section for imaging. From Figure 2B, it is clear that dcl5-1 has 
smaller number but larger sized tapetal cells, middle layer cells, and endothecial layer cells—
almost all the anther wall layers have larger sized and smaller numbered cells. Please clarify 
whether this applies to all dcl5-1 anthers growing in 28˚/22˚C or in the field. Are there any 
statistic data regarding this abnormality. 

2. This is just following my first question. If dcl5-1 mutants develop fewer but larger anther wall 
cells, it suggests a role before meiosis and likely on anther cell differentiation. In this case, it 
would make sense that the accumulation of 24nt phasiRNA in meiocytes and anthers (mainly 
tapetal cells) are parallel processes. The reduced number of binucleate tapetal cells in dcl5 mutant 
could be associated with less dynamic cell division. Are there any other changes that the authors 
have been notified such as changes of gene expression of cyclin or cyclin dependent kinase genes? 

3. Since this paper suggests a correlation between the temperature sensitivity of DCL5 to the 
agricultural belt of North America, a couple more sentences to introduce the origin of maize Hi-II 
line would be helpful for readers. In addition, it may be helpful to mention that it may vary among 
maize inbreds, especially for those with tropical origin, such as CML228. 

Reviewer #3 (Remarks to the Author): 

Nature Communications 
To Authors, 

This study shows that DCL5 (which is DCL3b) is required for anther wall developments and this 
dcl5 deficiency shows the temperature-sensitive male sterility through the decrease of 24-nt 
phasiRNAs in maize. However, it is already known that rice DCL3b, which is DCL5 in this paper, are 
required for 24-nt reproductive phasiRNA production in rice (Song et al., 2012). Furthermore, 
there are no correlation between 24-nt phasiRNAs expression and temperature-sensitive male 
sterility of dcl5 mutants under restrictive and permissive temperature. It remains unknown the 
mechanism of temperature-sensitive male sterility via 24nt-phasiRNAs and the function of 24 
reproductive phasiRNAs processed by DCL5. I would recommend against its publication in Nature 
Communications. 

Major comments 

1) DCL5 allele mutants 
There are no data of mendelian inheritance of “EACH” dcl5 allele (page 2 Line37). 

Please show these results including under 28/22℃, permissive and restrictive temperature. 

Please check whether there are other deletion or insertion regions in all mutants by whole genome 
sequences or other experiments, because there are 5 paralogues of DCLs in maize. 

Please show the expression of Dcl5 in all mutants. I couldn’t check the results of Fig S1. 

2) Female effect in dcl5 
How about female in dcl5 mutants? 

3) Line49-63 
Please explain the mono- and bi-nucleotide more detail with references. 

4) Dcl5 expression and DCL5 protein expression (line69-79) 
Please show DCL5 and DCL3a expression with in situ hybridization and show DCL5 and DCL3a 
protein localization in anther, because DCL3 is also important for 24-nt reproduction in dicots (Xia 
et al paper). 

5) 24-PHAS expression 
Please explain the difference of 24-PHAS expression in dcl5-4 and other three alleles (FigS8), 
though the expression of 24-nt phasiRNAs are decreased in all alleles (Fig3 and FigS7). 

6) 21-nt phasiRNAs/21PHAS 
Why were 21-nt phasiRNAs increased in all dcl5 mutants (FigS7) ? 
How about 21PHAS expression in mutants? 

7) 
There are no significant differences of “24nt phasiRNAs expression (also 24-PHAS)” under 
restrictive and permissive temperature (Fig3 and FigS8). Authors concluded that 24-nt phasiRNAs 
are dispensable at cool growing temperature for maize and may allow alternative pathways 
(line111,112, 113150). Please show the evidence. For example, the authors performed RNA-seq 
using WT and dcl5 under restrictive and permissive temperature. How about changing of genes of 
alternative pathways independent of 24nt phasiRNAs to support the authors interpretation? 

8) Targets of 24-nt phasiRNAs 
The authors concluded that characterization of downstream transcription, target, was 
uninformative (line 92). Please show the results. Because it seems that premeiotic phasiRNAs 
(21nt?) are used for searching target, not meiotic phasiRNAs in Zhei et al (2015) paper, which are 
referenced (line 93). 

9) It is recommended to fit this manuscript to Nature communications form.