Overexpression of the mutated EMB2279/SOT5 and EMB2654 CDS in WT leads to RDR6 dependent gene silencing
We previously reported that a weak allele of emb2279-2/sot5 mutants exhibits a virescent phenotype, which is caused by a point mutation that significantly reduces splicing efficiency of the seventh intron of SOT5 and generates two additional mRNA variants [5]. The smallest transcript that lacks 22-base pairs (bp) at the 3' end of the seventh exon is predicted to produce a truncated SOT5 with only 6 PPR motifs, named SOT5-m1 (Fig.1), whereas the largest transcript that contains the seventh intron is predicted to produce a mutated protein with 10 PPR motifs, named SOT5-m2 (Fig.1). To test whether the two predicted proteins is functional or not in plants, we cloned SOT5-m1 and SOT5-m2 CDS and transformed them under the control of the cauliflower mosaic virus 35S promoter into WT plants. We obtained 30 and 42 positive T1 transformants for 35S:SOT5-m1 and 35S:SOT5-m2 constructs, respectively. Our results showed that 80% of the 35S:SOT5-m1 transgenic lines exhibited severe leaf chlorosis at the early growth stage and these leaves were gradually turned into pale green later; and 48% of 35S:SOT5-m2 transgenic lines displayed relatively mild leaf chlorosis and virescence (Table1 and Fig.2a). This virescent phenotype was similar to that of the sot5 mutant. Then we choose several T1 transformants with chlorosis phenotype for gene expression analysis. Indeed, quantitative PCR analysis showed that the level of the endogenous SOT5 transcripts, detected by the specific primer pair spanning 5’UTR, was significantly decreased in 35S:SOT5-m1 transgenic line2 and line3 (less than 40% of WT) while the level of total SOT5 transcripts (mainly from the transgene), detected by the primer pair spanning intron 7 (Fig. 2b) or CDS (Fig. 2c) was significantly increased. Consistently, in 35S:SOT5-m2 transformants, the expression level of endogenous SOT5 was also significantly decreased (less than 60% of WT) and that of exogenous SOT5 was significantly increased (Fig. 2b-2d). Meanwhile, one of the SOT5 target gene, the plastid rpl2 gene showed the dramatic decrease of splicing efficiency in these transformants (Fig. 2b and 2e). In contrast, the splicing efficiency of plastid atpF gene (not the SOT5 target) was not decreased (Fig. 2b and 2e). These results indicate that overexpression of the mutated SOT5 CDS in WT leads to suppression of the endogenous gene expression, probably through the post transcriptional gene silencing (PTGS, also known as sense cosuppression) pathway.
To confirm this speculation, we transformed 35S:SOT5-m1 and 35S:SOT5-m2 constructs into the rdr6-11 mutant. RDR6 is a key component in the PTGS pathway and the rdr6-11 mutant presented elongated and curl downward leaves (Fig. 3a) [16]. It is expected that overexpression of homologous gene in this mutant is not able to trigger the PTGS pathway [17, 18]. Twenty-five 35S: SOT5-m1/rdr6 and eighteen 35S: SOT5-m2/rdr6 T1 transformants were obtained. As expected, none of the transformants exhibited chlorosis phenotype (Fig. 3a). RT-PCR analysis showed that expression levels of the transgene were significantly increased in the transformants, whereas the endogenous SOT5 was not decreased (Fig.3b-3d). Consequently, no splicing defects of plastid rpl2 was detected in these transformants (Fig. 3e). Taken together, our results indicate that overexpression of the mutated SOT5 CDS in the WT background leads to PTGS in a RDR6-dependent manner.
We then asked whether the above gene silencing could be repeated with other EMB PPR genes. To do this, we overexpressed a truncated CDS of EMB2654 (EMB2654-11M), into the WT background (Fig. 1). It has been reported that EMB2654, encoding a P family PPR protein was required for intron 1 splicing of the plastid gene rps12 [4]. As expected, all the 47 transgenic lines we screened for positive T1 transfromants exhibited leaf chlorosis (Table1 and Fig. 4a). RT-PCR analysis showed that expression of the endogenous EMB2654 was down-regulated (about 20% of WT) while the transgene was significantly overexpressed in these transformants (Fig. 4b-d). Indeed, the splicing efficiency of rps12 intron 1 was significantly decreased in the cosuppression lines, compared to that in WT (Fig. 4e). However, when the truncated EMB2654-11M CDS was overexpressed in rdr6-11, no leaf chlorosis was observed among 30 transformants (Fig. 4f). RT-PCR results showed that the endogenous EMB2654 was not decreased and the transgene of EMB2654 was strongly overexpressed in these transformants (Fig4g-4i). Consistently, the splicing efficiency of rps12 intron 1 was not decreased in these transformants (Fig. 4j). Thus, these results confirm that overexpression of the truncated EMB2654 CDS in the WT background leads to PTGS in a RDR6-dependent manner.
Functional analysis of EMB976 via its cosuppression lines
EMB976 is a functionally unknown P subfamily PPR protein, which contains 22 PPR motifs and is predicted to be localized in chloroplasts. Its knockout mutant has been demonstrated to be embryonically lethal [1]. To study its physiological role in plant growth, we constructed two plasmids, named EMB976-7M and EMB976-14M containing 7 and 14 PPR motifs (Fig.1), respectively, and transformed them into the WT background. Our results showed that four of eight 35S:EMB976-14M/Col-0 T1 transformants displayed yellowing young leaves (Fig. 5a and Table 1), whereas all of the thirteen 35S:EMB976-7M//Col-0 transformants had the same phenotype as WT (Table 1). To confirm whether gene silencing occurred in 35S:EMB976-14M/ Col-0, we checked expression levels of the endogenous EMB976 in the transformants with yellowing phenotype. As expected, the endogenous EMB976 was significantly down-regulated (less than 40% of WT) while the transgene EMB976 was significantly overexpressed in these transformants (Fig.5b and 5c), indicating that the phenotype of the transgenic lines is caused by the silencing of EMB976. Since the P family PPR proteins were often involved in organell RNA stability and splicing, we further examined the intron splicing of chloroplast genes in these 35S:EMB976-14M/ Col-0 cosuppression lines. Indeed, RT-PCR analysis showed that the precursors of ndhA, clpP1 intron 2 and ycf3 intron 1 were significantly and specifically over accumulated in these lines (Fig. 5d). q-RTPCR analysis furtherly showed that the splicing efficiency of clpP1 intron 2 and ycf3 intron 1 was significantly and specifically decreased in these lines when compared to that of ycf3 intron2 (Fig. 5e), suggesting chloroplast clpP1 intron 2 and ycf3 intron 1 were the possible targets of EMB976. However, further experiments are required for verification of the results. Again, no phenotypes were observed among the transformants when EMB976-14M was overexpressed in rdr6-11 (Fig. 5f). This result was consistent with that of RT-PCR analysis. In these transformants, the endogenous EMB976 transcripts was not downregulated while the transgene EMB976 was significantly overexpressed (Fig. 5g-5i). Consistently, the splicing efficiency of clpP1 intron 2 and ycf3 intron 1 was not decreased when compared to that of ycf3 intron2 (Fig. 5j). Thus, we conclude that gene silencing triggered by overexpession of truncated EMB genes is dependent on RDR6.
Table 1 The cosuppression phenotype and frequency of each construct when transformed into WT background
Construct name
|
Length of encoding protein (aa)
|
Number of PPR motifs
|
Phenotype of cosuppression
|
Number of total T1 transformants
|
Number of T1 transformants with visible chlorosis
|
Frequency of cosuppressiona
|
Severity of chlorosis in cosuppression lines
|
SOT5
|
978
|
11
|
yellowing inflorescence and cauline leaves
|
35
|
20
|
57%
|
very mild
|
SOT5-m1
|
712
|
6
|
albino young leaves at seedling stage
|
30
|
24
|
80%
|
strong
|
SOT5-m2
|
1006
|
10
|
partial albino leaves at seedling stage
|
42
|
20
|
48%
|
mild
|
EMB2654
|
822
|
18
|
ND
|
ND
|
ND
|
ND
|
ND
|
EMB2654-11M
|
550
|
11
|
chlorosis and variegated leaves at seedling stage
|
47
|
47
|
100%
|
mild
|
EMB976
|
1038
|
22
|
ND
|
ND
|
ND
|
ND
|
ND
|
EMB976-7M
|
443
|
7
|
WT-like
|
13
|
0
|
0%
|
ND
|
EMB976-14M
|
747
|
14
|
yellowing young leaves at seedling stage
|
8
|
4
|
50%
|
mild
|
a cosuppressed T1 tranfromants/total T1 transformants. ND: not determined.