Sugarcane mosaic caused by Sugarcane Mosaic Virus (SCMV) is one of the most important virus diseases of sugarcane. In the present study, changes in the transcription profile obtained by cDNA-AFLP analysis were investigated in two sugarcane varieties contrasting to SCMV resistance, when challenged with a severe virus strain. Healthy plants derived from meristem tip tissue culture were mechanically inoculated under greenhouse controlled conditions and sampled at 24, 48 and 72 hours after inoculation. A total of 392 transcript-derived fragments (TDFs) were verified in the resistant variety against 380 in the susceptible one. The two sugarcane genotypes showed differential behavior in the number of induced and repressed TDFs along the time-course samplings. Ten out of 23 sequenced TDFs (unique from the resistance variety), showed identity with known plant sequences, mostly related to plant defense mechanisms against pathogens. The cDNA-AFLP technique was effective in revealing changes in the transcription profile within and between contrasting varieties when challenged by SCMV.
The mosaic disease in different poaceous species can be caused by a subgroup of seven distinct members of family Potyviridae: Sugarcane Mosaic Virus (SCMV) which names the subgroup, Sorghum Mosaic Virus (SrMV), Maize Dwarf Mosaic Virus (MDMV), Johnson Grass Mosaic Virus (JGMV), Pennisetum Mosaic Virus (PenMV), Zea Mosaic Virus (ZeMV), belonging to the genus Potyvirus, and Sugarcane Streak Mosaic Virus (SCSMV) belonging to the new genus Poace Virus [
Despite the importance of mosaic to the crop, little is known about the transcript profile of sugarcane infected by SCMV. In the last years several approaches using molecular tools have become available to identify genes of interest in the development of new varieties. The cDNA-AFLP approach [
Two sugarcane varieties from the IAC Sugarcane Breeding Program, Brazil, IACSP95-5000 and IAC91-1099, respectively resistant and susceptible to SCMV, were obtained by meristem tip culture at the Sugarcane Bio factory of the IAC Sugarcane Research Centre (Ribeirão Preto, Brazil). The virus indexing of the micro-propa- gated plantlets was done by molecular diagnosis via RT-PCR with specific primers to the SCMV coat protein [
Experiments were conducted in an aphid proof green-house under natural sunlight, with environment temperatures varying from 22˚C - 34˚C, in a randomized block experimental design at the IAC Sugarcane Research Centre between January and March 2012. The sugarcane plantlets from meristem tip culture were acclimated in the green-house during 20 days and transplanted to 1 liter plastic vessels. Fifteen days after transplanting six plants of each variety at each of the three sampling time-points (24, 48 and 72 hours post inoculation—hpi) were mechanically inoculated using as source of inoculum Sorghum “Rio” leaves infected with SCMV Rib-1 [
The first leaf from the top to the bottom of the stalk with clearly visible dewlap (+1 leaf) of each replicate was individually collected from each treatment and respective controls and immediately stored in liquid nitrogen. Total RNA was extracted with Trizol reagent (Invitrogen, Carlsbad, USA) following the manufacturer’s instructions. The purity, integrity and quantification of the extracted RNA were evaluated through A260/A280 in a NanoDrop® (Promega, USA) along with electrophoresis in 1.5% agarose’s gel.
The cDNA-AFLP analysis was conducted in bulks of equal quantities of total RNA extracted from 3 plants chosen randomly out of the 6 replicates. The first and the second strands cDNA synthesis was performed with the RevertAidTM H Minus First Strand cDNA Synthesis Kit (Fermentas, Lithuania) following the manufacturer’s instructions. Two hundred nanograms of double strand cDNA were double digested with the restriction enzyme combinations EcoRI plus MseI and EcoRI plus MspI. The ligation of the adapters to the digested fragments, the pre-amplification and the selective amplification were performed as previously described [
The number of TDFs observed between the varieties across the sampling time points (24, 48 and 72 hpi) was compared by the Chi-Square test contingency table analysis (hpi and variety). To test the equality of the frequency of TDFs within each variety the Chi-Square test was applied assuming equal frequencies (1/3 of the total TFDs) across the sampling time points.
TDFs were excised from the gel, eluted in 50 μL of TE buffer (10 mM Tris-HCl, 1.0 mM EDTA, pH 8.0) and incubated overnight at 4˚C. TDFs were re-amplified with their respective selective primers as used in the cDNA-AFLP analysis and purified with the Wizard® SV Gel and PCR Clean Up System Kit, cloned into pGEM-T Easy (Promega) and sequenced using the BigDye® Terminator v3.1 Cycle Sequencing Kit. The nucleotide sequences were submitted to Basic Local Alignment Search Tool (BLAST) from NCBI [
One month after inoculation with SCMV Rib-1, plants from the susceptible variety (IAC91-1099) showed typical symptoms of infection, such as mosaic and reduced growth whereas plants from the resistant variety (IACSP95-5000) did not show any disease symptoms. The RT-PCR amplified a specific fragment of approximately 890 bp corresponding to the SCMV capsid protein in all inoculated plants, while no fragment was observed in the mock-inoculated ones (
The number of TDFs observed for each variety, only in the inoculated treatments (I), which probably corresponds to induced genes, as well the number of TDFs observed only in the non-inoculated controls (R), which may correspond to repressed genes in the inoculated treatments is showed for the respective 16 selective primer combinations (
Selective Primer Combination | Variety | Total | ||||||||||||
---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
IACSP95-5000 | IAC91-1099 | |||||||||||||
24 hpi | 48 hpi | 72 hpi | 24 hpi | 48 hpi | 72 hpi | |||||||||
I | R | I | R | I | R | I | R | I | R | I | R | |||
E-ACA/M1CTT | 5 | 8 | 9 | 7 | 4 | 4 | 8 | 5 | 6 | 6 | 2 | 4 | 68 | |
E-AAA/M1CTT | 7 | 8 | 7 | 8 | 6 | 3 | 0 | 4 | 6 | 7 | 6 | 4 | 66 | |
E-ACT/M1CTA | 4 | 5 | 4 | 6 | 3 | 6 | 6 | 4 | 4 | 7 | 6 | 4 | 59 | |
E-ACC/M1 CTT | 7 | 7 | 7 | 7 | 4 | 6 | 5 | 5 | 11 | 6 | 8 | 4 | 77 | |
E-AAG/M1CTT | 3 | 5 | 2 | 4 | 3 | 3 | 1 | 1 | 4 | 6 | 2 | 1 | 35 | |
E-ACT/M1CTT | 5 | 6 | 5 | 3 | 5 | 1 | 4 | 4 | 5 | 8 | 6 | 4 | 56 | |
E-ACT/M1CAA | 6 | 5 | 1 | 3 | 1 | 0 | 4 | 3 | 3 | 6 | 1 | 3 | 36 | |
E-AAC/M1CGT | 7 | 3 | 5 | 4 | 1 | 2 | 3 | 3 | 4 | 4 | 1 | 1 | 38 | |
E-AAG/M1CGT | 1 | 2 | 2 | 4 | 3 | 3 | 4 | 1 | 3 | 6 | 3 | 3 | 35 | |
E-AAC/M2ACT | 5 | 2 | 1 | 5 | 2 | 2 | 2 | 4 | 1 | 3 | 3 | 3 | 33 | |
E-AAC/M2TCG | 1 | 1 | 3 | 1 | 2 | 2 | 0 | 0 | 2 | 1 | 2 | 2 | 17 | |
E-ACG/M2ACT | 8 | 3 | 3 | 5 | 2 | 6 | 5 | 4 | 1 | 2 | 5 | 2 | 46 | |
E-AAC/M2GAA | 4 | 2 | 2 | 3 | 2 | 1 | 3 | 1 | 4 | 4 | 3 | 2 | 31 | |
E-AAC/M2ACA | 6 | 5 | 11 | 5 | 8 | 4 | 5 | 5 | 4 | 8 | 8 | 3 | 72 | |
E-ACG/M2TTG | 9 | 6 | 3 | 1 | 3 | 5 | 4 | 3 | 6 | 5 | 9 | 7 | 61 | |
E-AGC/M2ACA | 4 | 2 | 3 | 2 | 3 | 4 | 4 | 3 | 3 | 3 | 7 | 4 | 42 | |
Total (I/R) | 82 | 70 | 68 | 68 | 52 | 52 | 58 | 50 | 67 | 82 | 72 | 51 | 772 | |
Total (I) | 202 | 197 | 399 | |||||||||||
Total (R) | 190 | 183 | 373 | |||||||||||
Total/hpi | 152 | 136 | 104 | 108 | 149 | 123 | ||||||||
Total/Variety | 392 | 380 | 772 |
E: three-base-selective primer EcoRI (CTGCGTACCAATTC); M1: three-base-selective primer MseI (GATGAGTCCTGAGTAA); M2: three-base- selective primer MspI (GATGAGTCCTGATCGG).
The selective primer combination E-AAC/M2TCG produced the lowest number of TDFs (17), while the highest (77) was observed with the combination E-ACC/M1CTT. In overall, 772 TDFs were obtained, of which 392 observed in the resistant (IACSP95-5000) and 380 in the susceptible (IAC91-1099) variety. Three hundred and ninety nine (52%) out of the 772 TDFs, were specifically observed only during the plant-pathogen interaction, i.e., in the inoculated treatments.
The two varieties contrasting in their resistance to SCMV differed significantly in their expression profiles regarding the number of induced TDFs across the time points (χ2 = 7.29; P = 0.0262). This can be viewed by comparing the number of TDFs that may correspond to induced genes, along the different sampling time-points (
The identify transcripts that could be directly related to mosaic resistance, two situations were considered: 1) for each sampling time-point (24, 48 and 72 hpi) TDFs present exclusively in the resistant variety (IACSP95-5000) inoculated treatment, but absent in the controls as well as in the susceptible variety treatments (IAC91-1099) and respective controls, were selected; 2) for each sampling time-point, TDFs present either in the resistant variety controls or in the susceptible variety (treatments and controls), but absent in the resistant variety inoculated plants were selected (
(hpi) | Induced | Repressed |
---|---|---|
24 | 32 | 4 |
48 | 12 | 4 |
72 | 13 | 1 |
Total | 57 | 9 |
Out of the 57 differentially expressed fragments, exclusively found in the resistant variety (
In this study two varieties which differ in response to resistance to SCMV were investigated at the transcriptional level through the cDNA-AFLP technique when challenged by a virus severe strain. The use of plantlets cleaned by meristem-tip tissue culture and indexed by RT-PCR ensured that the experiment was carried out with virus-free plants, discarding the possibility of cross-protection, i.e., the occurrence of plant resistance to a virus strain induced by the previous systemic infection by a mild strain of the same virus species [
TDF1 | Selective primer combination2 | Fragment size (bp) | Accession No | Organism or gene | E-value3 | Query cover4 | Identity5 | ||
---|---|---|---|---|---|---|---|---|---|
24 hpi | |||||||||
45 | E-AAC/M1CGT | 100 | JN800037.1 | Retrotransposon complete sequence | 7.00E−12• | 53% | 93% | ||
52 | E-AAC/M2ACT | 300 | JX457396.1 | 23S ribosomal RNA (rrn23S) | 2.00E−60• | 79% | 100% | ||
63 | E-AAC/M2GAA | 330 | XM_002453856.1 | Hypothetical protein, mRNA | 7.00E−77• | 83% | 87% | ||
82 | E-ACG/M2ACT | 160 | XM_002444994.1 | Hypothetical protein, mRNA | 1.00E−51• | 83% | 93% | ||
48 hpi | |||||||||
29 | E-ACT/M1CTA | 220 | _ | None | _ | _ | _ | ||
41 | E-AAC/M1CGT | 450 | XP_002448166.1 | SORBIDRAFT_06g022385 | 1.00E−69† | 91% | 88% | ||
58 | E-AAC/M2TCG | 200 | XP_002467304.1 | SORBIDRAFT_01g024221 | 6.00E−08† | 37% | 84% | ||
78 | E-ACG/M2ACT | 350 | _ | None | _ | _ | _ | ||
72 hpi | |||||||||
14 | E-ACC/M1CTT | 280 | NP_001150511.1 | VQ motif family protein [Zea mays] | 1.00E−14† | 41% | 80% | ||
21 | E-ACT/M1CTT | 140 | XP_002455751.1 | SORBIDRAFT_03g023980 | 4.00E−14† | 47% | 65% | ||
28 | E-ACT/M1CTA | 300 | BT068454.2 | Zea mays full-length cDNA clone | 3.00E−31• | 31% | 92% | ||
34 | E-ACA/M1CTT | 290 | _ | None | _ | _ | _ | ||
81 | E-ACG/M2ACT | 180 | AE009947.2 | Saccharum hybrid cultivar SP-80-3280 chloroplast, complete genome | 4.00E−72• | 83% | 100% | ||
1TDF derived from the AFLP selective primer combination; 2E: EcoRI; M1: MseI; M2: MspI; 3E-value attributed to blast search, the best (lowest) Expect value of all alignments from the database sequence; 4Percentage of query covered by alignment to the database sequence; 5The highest percent identity of all query-subject alignments; •Alignment performed with the BlastN tool; †Alignment performed with BlastX tool.
symptoms were observed in all inoculated plants of the susceptible variety IAC91-1099. Moreover, once the absence or presence of the virus in the micro-propagated plantlets and in the treatments in all-time courses was confirmed by RT-PCR, it is expected that the TDFs herein disclosed by cDNA-AFLP reflect changes exclusively derived from the plant-virus interaction in the transcription profiles within and between the contrasting sugarcane varieties. According to [
In our work, polymorphism obtained by digestion with the combination of the restriction enzymes EcoRI/MseI and EcoRI/MspI was explored using primers with three selective bases. As pointed out by [
The resistant variety presented a very different expression profile in comparison to the susceptible one, particularly in relation to induction TDFs over the time-course samplings after inoculation. Similar results were also reported by [
According to [
The suitability of the experimental design and of the cDNA AFLP technique for these analysis offers the possibility of its application for the generation and sequencing of a greater number of TDFs, which will allow not only a better understanding of the resistance mechanisms involved in SCMV infection, but also the identification of candidate genes to be applied in the development of molecular markers aiming the genetic mapping for virus resistance in sugarcane.
This research was supported by the São Paulo Research Foundation (FAPESP) Project BIOEN 2008/56146-5 and Instituto Agronômico de Campinas (IAC). C. N. F. Medeiros was a recipient of a Master’s fellowship from FAPESP (2012/15060-6).