Molecular Characterization of Sugarcane Yellow Leaf Virus (SCYLV) and its Effect on Sucrose Transporters in Sugarcane Saccharum spp. hybrids
Abdelaleim Ismail Ibrahim ElSayed
- Sugarcane is an important crop plant and has served as a source of sugar for hundreds of years, recently it is used to produce bioethanol, a renewable bio-fuel energy source. Sugarcane yellow leaf virus (SCYLV) was detected in the late 1990s first in Hawaii as a causal agent of a sugarcane disease which leads to sugarcane yellow leaf syndrome and reduced sugar yield. The presence of Sugarcane yellow leaf virus was determined by RT-PCR in several sugarcane cultivars, mostly from Hawaii. Interesting was the comparison of so-called susceptible versus resistant cultivars. As expected, the susceptible Hawaiian cultivars H73-6110 and H87-4094 showed strong PCR amplification products of SCYLV, while the virus-free line H87-4094, produced by tissue culture, showed no PCR product. The three resistant cultivars H87-4319, H78-4153 and H78-7750 showed quite different amplification patterns. While H78-4153 and H78-7750 expressed a weak but specific band of the correct size, unexpectedly H87-4319 showed strong amplification product. Three Cuban cultivars (C1051-73, JA-605 and CP52-43) showed low titer of SCYLV. No PCR amplificate was obtained with the moderately susceptible cultivar H65-7052. Aphids feeding on cv. H87-4094 contained sufficient virus to yield a SCYLV-signal similar in strength as from preparations from resistant cultivars. Northern blot analysis supported the results obtained from RT-PCR. The presence of SCYLV in the cultivars with low amount of virus titer (H87-4319, H78-7750 and H78-4153) indicated that they should better be called tolerant for the virus in the sense that they allow a low replication rate for SCYLV. Virus preparations from 3 Hawaiian cultivars (two susceptible and one resistant) were fully sequenced. Quantitative analysis for four different genome regions of SCYLV covering the 6 ORFs has been performed for these 3 cultivars using the GeXP analysis system. The transcript levels of the different regions of SCYLV in these cultivars were present at very different quantities, for example ORF0-1 transcripts were up to 10 times more frequent than transcripts of ORF3-4. The SCYLV-sequences from the 3 Hawaiian cultivars were aligned to published full and partial sequences. The phylograms corroborated previous findings that the so-called YLS-segment coding for the coat protein shows the least genetic diversity, whereas the other sequence fragments A-D, representing the ORFs 0-5, expressed a twofold higher diversity. The phylograms of partial sequences and of the whole genome placed the Hawaiian SCYLV-strains next to the Peru strain, apart from the BRA-strains and well apart from the REU-strains. It is proposed that the Hawaiian SCYLV is considered as own group together with the Peru strain as HAW-PER. The sequences from the two susceptible cultivars had a deletion of 48 to 54 nt in ORF1, which codes for the gene silencing suppressor/RNA-dependent RNA-polymerase complex. It is speculated that this deletion is important for the proliferation rate of the virus in the plant. Sucrose is the main product of sugarcane, which accumulates in the stalk internodes in excess of 50 % of the dry weight. To gain an overview of the physiological status of SCYLV-infected sugarcane compared to virus-free plants, gene expression. Sucrose increased rapidly between internodes 3 and 7, reaching a maximum in internodes 7. Sugars content in leaves, seedling and internodes were increased as effect of the SCYLV-infection. Sucrose phosphate synthase (SPSII) transcript levels were approximately the same in sink, source and internodes with a trend to be higher in the mature internodes. A sucrose transporter of Hawaiian cultivar was isolated and sequenced and classified as ShSUT1A. There is high variability among the SUT1 subfamily with identities of 70-97%. The identity between ShSUT1A and ShSUT1 was 97.4%. It is expressed in sink, source and storage tissues. The ShSUT1A was expressed at approximately similar extent in SCYLV-infected and virus-free sugarcane. In addition a partial sequence of a sucrose transporter belonging to the SUT4 family was first obtained in sugarcane and its transcript levels in plant organs were measured. Quantitative analysis for sucrose transporters (ShSUT1 and ShSUT4) using the GeXP analysis system showed that sucrose transporter ShSUT1 was at a higher transcript expression than ShSUT4 in sink and source leaves, but not in mature internodes. In conclusion, - SCYLV from Hawaiian cultivars was characterized as belonging to an own subgroup (HAW- PER), - A deletion of 48-54 nt was detected in the SCYLV-sequence from susceptible cultivars, which may be correlated to virus proliferation, and - large differences in transcript levels of the viral ORFs were found. - Sucrose transporter transcripts and SPSII transcripts were not strictly correlated to SCYLV- infection and do not explain the pathological effect of SCYLV on sugarcane.