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



Reviewers' comments: 

Reviewer #1 (Remarks to the Author): 

This manuscript, entitled "Dicer-like 5 deficiency confers temperature-sensitive male sterility in 
maize" reports the involvement of DCL5 in 24-nt phasiRNA biogenesis in maize and shows that 
DCL5 and 24-nt phasiRNAs play vital roles in tapetal functions at high temperatures. In general, 
the findings are very interesting. However, I still have some comments. 

1.If DCL5 and 24-nt phasiRNAs are only indispensable under higher temperature, but not required 
for anther development under cooler temperatures, one would expect to see increased expression 
of DCL5 in the wild-type anthers under higher temperature. Results shown in Fig. S2 are from dcl5 
mutant anthers under permissive and restrictive temperature, which does not explain whether 
DCL5 and 24-nt phasiRNA respond to heat treatment in the wild-type. 
2.The impacts of DCL5 on the tapetum are obvious, and the tapetum is the primary site of 24-nt 
biogenesis. It has previously been shown that tapetal cells play several important roles during 
male reproduction, such as supporting meiocytes, secreting enzymes to digest callose walls, 
mediating microscope development, and depositing components for pollen coats. Since the 
tapetum layer has multiple functions, it is necessary to determine what stage of gametogenesis is 
affected. Only showing abnormal tapetum morphology is a bit disappointing. Moreover, for 
example, if callose degradation is impaired in the dcl5 mutant, it would support the authors’ 
speculation that 24-nt phasiRNAs are essential for tapetal redifferentiation for biosynthesis and 
secretion of materials that support haploid microgametophytes (lines 186 and 187). 
3.Were tapetum cells of the dcl5 mutant binucleate under permissive temperate? 
4.In the rice tip2 mutant in which expression of DCL5 and production of 24-nt phasiRNA are 
severely affected (PLoS Genet, 14(2):e1007238), 5-7% of male meiosis showed segregation 
defects. Is it possible that the maize dcl5 mutant exhibits mild meiotic abnormality? 
5.In the comparison of Figs. 2C and 2D, nuclei in Fig. 2C (dcl5 mutant?) are larger and have a 
higher percentage of binucleate tapetal cells than nuclei shown in Fig 2D (wild-type). This is not 
consistent with what is stated in the main text. Please clarify. 
6.Line 445: In the figure legend, the orange boxes mark “two” binucleate tapetal cells. However, I 
only see one tapetal cell in the orange box. 
7.Although the authors state that “In extensive confocal microscopic examination of dcl5-1 anthers, 
we observed no significant or consistent differences in cell number or volume compared to fertile 
siblings” (lines 91-92). However, by examining Fig. S8, I feel it is very difficult to make such a 
conclusion. Statistical analysis is required to support this point. 
8.The scale bar in Fig. 4D-4I may be incorrect. 

Reviewer #3 (Remarks to the Author): 

Nature Communications 
To Authors, 

This revise is getting better than before, but there were parts that the authors do not answer to 
my questions. Though authors claim that 24-nt phasiRNAs are dispensable at cooler temperatures, 
more experimental evidence should be shown towards this statement. Because there is no 
correlation between 24-nt phasiRNAs expression and temperature-sensitive male sterility of dcl5 
mutants under restrictive and permissive temperature. If this revise is included in the “24-nt 
reproductive phasiRNAs are broadly present in angiosperm”, not back to back, the data and 
conclusion are reasonable to Nature Communications. 

1) RNA/small RNA-sequencing using “WT or control” under permissive and restrictive temperature 
conditions seem to be important to reveal the expression of DCL5, phasiRNAs and 24-PHAS under 

high and low conditions. 

2) Mendelian inheritance of dcl5-1 (Sup table1) 
Please show mendelian inheritance analysis using backcross segregants (N>50), because the 
authors produced the BC1 to 3 of dcl5-1 (Sup table 1). 

3) Sup figure 10 
In this revise version, the abundance pattern in dcl5 mutants in SupFig10 is different from that of 
previous same figure (Sup fig8 in previous version), especially dcl5-1 permissive and restrictive 
seems to be opposite. Which figure is true?