Supplementary Information Virus-induced gene complementation reveals a transcription factor network in modulation of tomato fruit ripening Tao Zhou 2,3, Hang Zhang 2,4, Tongfei Lai 1, Cheng Qin 1, Nongnong Shi 1, Huizhong, Wang 1, Mingfei Jin 2,5, Silin Zhong 6, Zaifeng Fan 3, Yule Liu 7, Zirong Wu 5, Stephen Jackson 2, James J Giovannoni 6, Dominique Rolin 8, Philippe Gallusci 8, and Yiguo Hong 1,2* 1 Research Centre for Plant RNA Signalling, College of Life and Environmental Sciences, Hangzhou Normal University, Hangzhou 310036, China. 2 Warwick HRI and School of Life Science, University of Warwick, Warwick CV35 9EF, UK. 3 Department of Plant Pathology and State Key Laboratory of Agrobiotechnology, China Agricultural University, Beijing 100094, China. 4 Chengdu Rongsheng Pharmaceuticals, Chengdu 610041, China. 5 School of Life Science, East China Normal University, Shanghai 200062, China. 6 Boyce Thompson Institute for Plant Science Research, Cornell University, Ithaca, New York 14853-2901, USA. 7 School of Life Sciences, Tsinghua University, Beijing 100084, China. 8 Université de Bordeaux 1 UMR 1332, Biologie du Fruit et Pathologie, 33883 Bordeaux, France. * Correspondence should be addressed to Y. H. (yiguo.hong@hznu.edu.cn, yghongabc@googlemail.com or yiguo.hong@warwick.ac.uk) These authors contributed equally to this work. 1
Supplementary Text Stability of LeMADS-RIN RNA in VIGC vectors. When amplified by RT-PCR with a pair of primers PP82 and PP83 flanking the inserted LeMADS-RIN RNA in PVX/LeMADS-RIN, PVX/mLeMADS-RIN, PVX/HisG::LeMADS-RIN and PVX/HisG::mLeMADS-RIN, an intact construct should yield a single band (~1000-nt PVX-RIN RNA, Fig. S3), and any shorter fragments [e.g. ~700-nt PVX-RIN RNA (partial deletion) and 258-nt PVX RNA without RIN insert, Fig. S3] would indicate that part of the LeMADS-RIN RNA sequence was lost in these PVX-based VIGC vectors. PVX with a shorter version of the LeMADS-RIN RNA or PVX without any foreign sequence would be expected to replicate more efficiently. Consequently the accumulation of the RT-PCR products corresponding to the deleted-versions (partial or total deletion) of viruses should go up, while the full-size insert band originated from the non-deletion VIGC vectors should diminish. However, RT-PCR analysis showed a completely different situation, with two smaller bands present in considerably less abundance in all samples (Fig. S3). These data suggest that the full-length LeMADS-RIN RNA was largely maintained in all VIGC vectors during the course of infection and virus-induced complementation in rin fruits. However partial or complete deletion of the LeMADS-RIN RNA from a small portion of viral genomic RNAs of all VIGC vectors had taken place. The precise mechanism about how the LeMADS-RIN RNA was deleted from recombinant viruses is unknown. It is possible that such deletion could occur through homologous RNA recombination between the duplicated viral coat protein subgenomic promoter sequences, even between the endogenous rin transcript (which is transcribed from tomato deletion mutant rin gene) and the LeMADS- RIN RNA in the VIGC vectors. Taken together, these data demonstrate that the LeMADS- 2
RIN RNA was stable in the PVX-based VIGC vectors, consistent with its function to complement rin mutant fruits (Fig. 2). Supplementary Figure Legends Figure S1 Map of PVX and PVX/HisGMF1. A double-stranded oligonucleotide fragment which codes an oligopeptide of six histidines and one glycine (His6-Gly) was obtained by annealing a pair of primers PP645 and PP646, and cloned into the ClaI site of the PVX vector 28 to produce PVX/HisGMF1. Viral RNA dependent RNA polymerase (166K), movement proteins (25, 12 and 8K) and coat protein (CP) as well as the His6-Gly tag are indicated. Unique cloning restriction enzymes, T7 and T3 promoters as well as the SpeI site at the end of the PVX genome are also indicated. Nucleotides are numbered according to the original PVX vector in pbluescript 28. Figure S2 sqrt-pcr assays of viral transient expression of LeMADS-RIN mrna from VIGC vectors in rin fruits. Total RNA was extracted from two mock-injected rin fruits (I and II) or from fruits injected with PVX/LeMADS-RIN, PVX/meLeMADS-RIN or PVX/HisG::mLeMADS-RIN at 3 wpi and used for sqrt-pcr with 30 cycles of amplification. RNA samples from the ripe red sectors (R) and non-ripe green sectors (G) of two fruits (I and II) injected with PVX/LeMADS-RIN were analysed. Equal amount of total RNA (100 ng) used in each of the sqrt-pcr reactions was verified by detecting a similar level of 18S rrna in all samples. The positions and sizes of the 1-kb DNA marker (Ladder) and positions of RT-PCR products specific for PVX::LeMADS-RIN mrna (PVX- RIN RNA) as well as 18S rrna are indicated. 3
Figure S3 Stability of LeMADS-RIN RNA in VIGC vectors. Total RNA extracted from rin fruits injected with PVX, PVX/LeMADS-RIN, PVX/meLeMADS-RIN, PVX/HisG::LeMADS-RIN or PVX/HisG::mLeMADS-RIN at 3 weeks post-injection was used for sqrt-pcr with 30 cycles of amplification using a pair of PVX genomic primers PP82 and PP83 (Table S1; Table S2). This pair of primers allows amplifying a 258 bp fragment from the PVX genome harbouring no foreign RNA insert. Three RT-PCR products which represented PVX::LeMADS-RIN (PVX-RIN) RNA of the predicted size of approximately 1000 nucleotides (nt), PVX::LeMADS-RIN RNA with partial deletion of about 700 nt and 258-nt PVX RNA without any LeMADS-RIN insert were detected. Relative levels of accumulation of VIGC viruses with partial or complete deletion of the LeMADS-RIN RNA were estimated to be less than 1% or 1 5% of that of VIGC viruses having the full-length LeMADS-RIN RNA, respectively, when comparing the densities of corresponding ethidium bromide-stained RT-PCR bands in each sample. The positions and sizes of the 1-kb DNA marker (Ladder) and positions of three RT-PCR products as well as 18S rrna are indicated. 4
Supplementary Tables Supplementary Table S1 Sequences of Primers used in this study Primer Sequences (5-3 ) Origin PP82 CAGTGTTGGCTTGCAAACTAG PVX PP83 TGGCAGGAGTTGCGCCTGCAG PVX PP271 CGGCTACCACATCCAAGGAAGG 18S rrna PP272 GAGCTGGAATTACCGCGGCTG 18S rrna PP298 CCTCACATCGATGGAAACTAACAAATGGGAAGGGA LeSPL-CNR PP300 TAACAGCGGCCGTCTGCTACATTGCTGACAGAATC LeSPL-CNR PP402 AGTTGAATCGATGGGATCTGGGCATATATTTTTC LeHB1 PP403 AAATTGGGCCCAGACCAGAACCATCCAATAGGCTC LeHB1 PP404 GCTCATATCGATGGTGTCGATTGAACAAGTAGA SlTPR1 PP405 AAACCCGGGCCCCTTCTGAAATTGAACAGAGTAG SlTPR1 PP797 TCTTCAATCGATATGGGTAGAGGGAAAGTAGA LeMADS-RIN (start codon) PP798 TCTTCAATCGATTAGGGTAGAGGGAAAGTAGA LeMADS-RIN (mutated start codon) PP799 ACTCCACGGCCGAAGCATCCATCCAGGTACAAC LeMADS-RIN 5
Supplementary Table S2 Primers for cloning and RT-PCR detection Primer set RT-PCR Cloning PP82/PP83 PP82/PP799 PP271/PP272 PP298/PP300 PP402/PP403 PP404/PP405 PVX, PVX-RIN RNA PVX-RIN RNA 18S rrna LeSPL-CNR mrna LeHB1 mrna SlTPR1 mrna PP797/PP799 PP797/PP799 PP798/PP799 PP798/PP799 PVX/LeMADS-RIN PVX/HisG::LeMADS-RIN PVX/mLeMADS-RIN PVX/HisG::mLeMADS-RIN 6