Limits...
Identification of Hop stunt viroid infecting Citrus limon in China using small RNAs deep sequencing approach.

Su X, Fu S, Qian Y, Xu Y, Zhou X - Virol. J. (2015)

Bottom Line: A bias of HSVd-siRNAs toward sequences beginning with a 5'-Guanine was observed.Furthermore, hotspot analysis showed that a large amount of HSVd-siRNAs derived from the central and variant domains of the HSVd genome.Our results suggest that C. limon could set up a small RNA-mediated gene silencing response to Hop stunt viroid, Interestingly, based on bioinformatics analysis, our results also suggest that the large amounts of HSVd-siRNAs from central and variant domains might be involved in interference with host gene expression and affect symptom development.

View Article: PubMed Central - PubMed

Affiliation: Institute of Biotechnology, State Key Laboratory of Rice Biology, Zhejiang University, Hangzhou, 310029, People's Republic of China. suxiu@zafu.edu.cn.

ABSTRACT

Background: The advent of next generation sequencing technology has allowed for significant advances in plant virus discovery, particularly for identification of covert viruses and previously undescribed viruses. The Citrus limon Burm. f. (C. limon) is a small evergreen tree native to Asia, and . China is the world's top lemon-producing nation.

Findings: In this work, lemon samples were collected from southwestern of China, where an unknown disease outbreak had caused huge losses in the lemon production industry. Using high-throughput pyrosequencing and the assembly of small RNAs, we showed that the Hop stunt viroid (HSVd) was present in C. limon leaf sample. The majority of it is a main lemon producing agricultural cultivarHop stunt viroid derived siRNAs (HSVd-siRNAs) in C. limon were 21 nucleotides in length, and nearly equal amount of HSVd-siRNAs originated from the plus-genomic RNA strand as from the complementary strand. A bias of HSVd-siRNAs toward sequences beginning with a 5'-Guanine was observed. Furthermore, hotspot analysis showed that a large amount of HSVd-siRNAs derived from the central and variant domains of the HSVd genome.

Conclusions: Our results suggest that C. limon could set up a small RNA-mediated gene silencing response to Hop stunt viroid, Interestingly, based on bioinformatics analysis, our results also suggest that the large amounts of HSVd-siRNAs from central and variant domains might be involved in interference with host gene expression and affect symptom development.

No MeSH data available.


Related in: MedlinePlus

Reverse transcriptase PCR amplification of HSVd with a specific back-to-back primer. Lane 1, DNA ladder; Lane 2, healthy control; Lane 3, HSVd infected Citrus limon sample
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
getmorefigures.php?uid=PMC4492010&req=5

Fig2: Reverse transcriptase PCR amplification of HSVd with a specific back-to-back primer. Lane 1, DNA ladder; Lane 2, healthy control; Lane 3, HSVd infected Citrus limon sample

Mentions: C. limon leaf samples were collected in September 2012 from a lemon orchard in the city of DeHong, Yunnan province, China. The C. limon tree displayed stunting, leaf roll and mottle symptoms (Fig. 1), along with poor yield. Total RNAs were extracted using Trizol Reagent following the manufacturer’s instructions (Invitrogen, CA, USA). Total small RNAs ranging from 18 to 28 nucleotides (nt) were excised from 15 % polyacrylamide gel (PAGE) for ligation to 3′ and 5′ adaptors. After purification by electrophoresis, the final ligation products were reverse-transcribed and a cDNA library was constructed. After sequencing and trimming the adaptor sequences, 18–28 nt short reads were collected. The velvet program was chosen for genome assembly with 17 nucleotides as the minimal overlapping length (k-mer) required for joining two siRNAs into a contiguous sequence (contig) [5–7]. Assembly of 1.76 million vsiRNAs yielded 2613 contigs, including 535 contigs with lengths above 80 nt and 2078 with lengths below 80 nt. These assembled contigs were then aligned with the BLASTN program using the standard parameters in genome assembly (contigs with ≥90 % similarity). One long contig (294-nt), homologous to the nucleotide sequence of Hop stunt viroid-citrus strain with 100 % similarity (Accession No. FJ716188) was detected. This contig covered 97.35 % of the whole HSVd genome, lacking only the first 10 nucleotides. Subsequently, the existence of an intact HSVd genome in C. limon was verified by reverse transcriptase PCR using a pair of back-to-back primers (Fig. 2). (Forward primer: 5′-CCAACCTGCTTTTTGTCTATCTGAG-3′ and reverse primer: 5′-AAGACGAACCGAGAGGTGATGC-3′). The integrity of the amplified genome was verified by sequencing, and we found that the whole genome of our HSVd shared 100 % similarity with the HSVd CC-D isolate 1 (Accession No. FJ716188).Fig. 1


Identification of Hop stunt viroid infecting Citrus limon in China using small RNAs deep sequencing approach.

Su X, Fu S, Qian Y, Xu Y, Zhou X - Virol. J. (2015)

Reverse transcriptase PCR amplification of HSVd with a specific back-to-back primer. Lane 1, DNA ladder; Lane 2, healthy control; Lane 3, HSVd infected Citrus limon sample
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4492010&req=5

Fig2: Reverse transcriptase PCR amplification of HSVd with a specific back-to-back primer. Lane 1, DNA ladder; Lane 2, healthy control; Lane 3, HSVd infected Citrus limon sample
Mentions: C. limon leaf samples were collected in September 2012 from a lemon orchard in the city of DeHong, Yunnan province, China. The C. limon tree displayed stunting, leaf roll and mottle symptoms (Fig. 1), along with poor yield. Total RNAs were extracted using Trizol Reagent following the manufacturer’s instructions (Invitrogen, CA, USA). Total small RNAs ranging from 18 to 28 nucleotides (nt) were excised from 15 % polyacrylamide gel (PAGE) for ligation to 3′ and 5′ adaptors. After purification by electrophoresis, the final ligation products were reverse-transcribed and a cDNA library was constructed. After sequencing and trimming the adaptor sequences, 18–28 nt short reads were collected. The velvet program was chosen for genome assembly with 17 nucleotides as the minimal overlapping length (k-mer) required for joining two siRNAs into a contiguous sequence (contig) [5–7]. Assembly of 1.76 million vsiRNAs yielded 2613 contigs, including 535 contigs with lengths above 80 nt and 2078 with lengths below 80 nt. These assembled contigs were then aligned with the BLASTN program using the standard parameters in genome assembly (contigs with ≥90 % similarity). One long contig (294-nt), homologous to the nucleotide sequence of Hop stunt viroid-citrus strain with 100 % similarity (Accession No. FJ716188) was detected. This contig covered 97.35 % of the whole HSVd genome, lacking only the first 10 nucleotides. Subsequently, the existence of an intact HSVd genome in C. limon was verified by reverse transcriptase PCR using a pair of back-to-back primers (Fig. 2). (Forward primer: 5′-CCAACCTGCTTTTTGTCTATCTGAG-3′ and reverse primer: 5′-AAGACGAACCGAGAGGTGATGC-3′). The integrity of the amplified genome was verified by sequencing, and we found that the whole genome of our HSVd shared 100 % similarity with the HSVd CC-D isolate 1 (Accession No. FJ716188).Fig. 1

Bottom Line: A bias of HSVd-siRNAs toward sequences beginning with a 5'-Guanine was observed.Furthermore, hotspot analysis showed that a large amount of HSVd-siRNAs derived from the central and variant domains of the HSVd genome.Our results suggest that C. limon could set up a small RNA-mediated gene silencing response to Hop stunt viroid, Interestingly, based on bioinformatics analysis, our results also suggest that the large amounts of HSVd-siRNAs from central and variant domains might be involved in interference with host gene expression and affect symptom development.

View Article: PubMed Central - PubMed

Affiliation: Institute of Biotechnology, State Key Laboratory of Rice Biology, Zhejiang University, Hangzhou, 310029, People's Republic of China. suxiu@zafu.edu.cn.

ABSTRACT

Background: The advent of next generation sequencing technology has allowed for significant advances in plant virus discovery, particularly for identification of covert viruses and previously undescribed viruses. The Citrus limon Burm. f. (C. limon) is a small evergreen tree native to Asia, and . China is the world's top lemon-producing nation.

Findings: In this work, lemon samples were collected from southwestern of China, where an unknown disease outbreak had caused huge losses in the lemon production industry. Using high-throughput pyrosequencing and the assembly of small RNAs, we showed that the Hop stunt viroid (HSVd) was present in C. limon leaf sample. The majority of it is a main lemon producing agricultural cultivarHop stunt viroid derived siRNAs (HSVd-siRNAs) in C. limon were 21 nucleotides in length, and nearly equal amount of HSVd-siRNAs originated from the plus-genomic RNA strand as from the complementary strand. A bias of HSVd-siRNAs toward sequences beginning with a 5'-Guanine was observed. Furthermore, hotspot analysis showed that a large amount of HSVd-siRNAs derived from the central and variant domains of the HSVd genome.

Conclusions: Our results suggest that C. limon could set up a small RNA-mediated gene silencing response to Hop stunt viroid, Interestingly, based on bioinformatics analysis, our results also suggest that the large amounts of HSVd-siRNAs from central and variant domains might be involved in interference with host gene expression and affect symptom development.

No MeSH data available.


Related in: MedlinePlus