San-Ji Gao

Enhanced Transgene Expression in Sugarcane by Co-Expression of Virus-Encoded RNA Silencing Suppressors

Post-transcriptional gene silencing is commonly observed in polyploid species and often poses a major limitation to plant improvement via biotechnology. Five plant viral suppressors of RNA silencing were evaluated for their ability to counteract gene silencing and enhance the expression of the Enhanced Yellow Fluorescent Protein (EYFP) or the β-glucuronidase (GUS) reporter gene in sugarcane, a major sugar and biomass producing polyploid. Functionality of these suppressors was first verified in Nicotiana benthamiana and onion epidermal cells, and later tested by transient expression in sugarcane young leaf segments and protoplasts. In young leaf segments co-expressing a suppressor, EYFP reached its maximum expression at 48–96 h post-DNA introduction and maintained its peak expression for a longer time compared with that in the absence of a suppressor. Among the five suppressors, Tomato bushy stunt virus-encoded P19 and Barley stripe mosaic virus-encoded γb were the most efficient. Co-expression with P19 and γb enhanced EYFP expression 4.6-fold and 3.6-fold in young leaf segments, and GUS activity 2.3-fold and 2.4-fold in protoplasts compared with those in the absence of a suppressor, respectively. In transgenic sugarcane, co-expression of GUS and P19 suppressor showed the highest accumulation of GUS levels with an average of 2.7-fold more than when GUS was expressed alone, with no detrimental phenotypic effects. The two established transient expression assays, based on young leaf segments and protoplasts, and confirmed by stable transgene expression, offer a rapid versatile system to verify the efficiency of RNA silencing suppressors that proved to be valuable in enhancing and stabilizing transgene expression in sugarcane.

A One-Step Real-Time RT-PCR Assay for the Detection and Quantitation of Sugarcane Streak Mosaic Virus

Sugarcane mosaic disease is caused by the Sugarcane streak mosaic virus (SCSMV; genus Poacevirus, family Potyviridae) which is common in some Asian countries. Here, we established a protocol of a one-step real-time quantitative reverse transcription PCR (real-time qRT-PCR) using the TaqMan probe for the detection of SCSMV in sugarcane. Primers and probes were designed within the conserved region of the SCSMV coat protein (CP) gene sequences. Standard single-stranded RNA (ssRNA) generated by PCR-based gene transcripts of recombinant pGEM-CP plasmid in vitro and total RNA extracted from SCSMV-infected sugarcane were used as templates of qRT-PCR. We further performed a sensitivity assay to show that the detection limit of the assay was 100 copies of ssRNA and 2 pg of total RNA with good reproducibility. The values obtained were approximately 100-fold more sensitive than those of the conventional RT-PCR. A higher incidence (68.6%) of SCSMV infection was detected by qRT-PCR than that (48.6%) with conventional RT-PCR in samples showing mosaic symptoms. SCSMV-free samples were verified by infection with Sugarcane mosaic virus (SCMV) or Sorghum mosaic virus (SrMV) or a combination of both. The developed qRT-PCR assay may become an alternative molecular tool for an economical, rapid, and efficient detection and quantification of SCSMV.

Quick detection of Leifsonia xyli subsp. xyli by PCR and nucleotide sequence analysis of PCR amplicons from Chinese Leifsonia xyli subsp. xyli isolates

A quick polymerase chain reaction (PCR) assay was developed for the detection of Leifsonia xyli subsp. xyli (Lxx), the bacterial causal agent of ratoon stunting disease (RSD) of sugarcane, in xylem sap samples from stalks. After removal of abiotic impurities and large molecular weight microorganisms by a 3000 rpm, 5 min centrifugation, the Lxx bacteria were precipitated from the xylem sap by a 12,000 rpm, 10 min centrifugation. The Lxx cells were re-suspended in 50 μl freshly prepared Buffer A (0.1M NaOH + 2% Tween 20), lysed by heating at 95°C for 10 min, cooled down on ice for 3 min, and neutralized by mixing with 50 μl of freshly prepared Buffer B (0.1M Tris-HCl, pH 8.0 + 2 mM EDTA). The resulting lysates were directly subjected to conventional PCR with Lxx-specific primers. Of the 31 varieties tested, 19 were positive for Lxx infection, including all FN, GT, and YT varieties. These varieties also tested positive by PCR on DNA templates extracted from xylem sap samples using a CTAB procedure and by two enzyme-immunoassays, dot-blot (DBEIA) and direct antigen coating (DAC-ELISA). DB-EIA and DAC-ELISA detected Lxx in 90.3 and 93.5%, respectively, of the varieties while the detection rate with PCR was 61.3%. The modified PCR assay was quick and more economical. It did not require organic solvent usage or ethanol precipitation, but produced the same level of detection as that of the PCR using DNA samples prepared by the CTAB procedure. All the PCR amplicons were 439 bp in size sharing the same nucleotide sequence. This consensus sequence, GenBank Accession EU723209, aligned perfectly with three other Lxx nucleotide sequences available in the GenBank, AE016822, AF034641, and DQ232616. However, two mis-matches, a (G?A) transversion and a deletion, were found in another nucleotide sequence AF056003.

Rapid and Quantitative Detection of Leifsonia xyli subsp. xyli in Sugarcane Stalk Juice Using a Real-Time Fluorescent (TaqMan) PCR Assay

Ratoon stunting disease (RSD) of sugarcane, one of the most important diseases seriously affecting the productivity of sugarcane crops, was caused by the bacterial agent Leifsonia xyli subsp. xyli (Lxx). A TaqMan probe-based real-time quantitative polymerase chain reaction (qPCR) assay was established in this study for the quantification of Lxx detection in sugarcane stalk juice. A pair of PCR primers (Pat1-QF/Pat1-QR) and a fluorogenic probe (Pat1-QP) targeting the Part1 gene of Lxx were used for the qPCR assay. The assay had a detection limit of 100 copies of plasmid DNA and 100 fg of Lxx genomic DNA, which was 100-fold more sensitive than the conventional PCR. Fifty (28.7%) of 174 stalk juice samples from two field trials were tested to be positive by qPCR assay, whereas, by conventional PCR, only 12.1% (21/174) were tested to be positive with a published primer pair CxxITSf#5/CxxITSr#5 and 15.5% (27/174) were tested to be positive with a newly designed primer pair Pat1-F2/Pat1-R2. The new qPCR assay can be used as an alternative to current diagnostic methods for Lxx, especially when dealing with certificating a large number of healthy cane seedlings and determining disease incidence accurately in commercial fields.

Identification and characterization of Xanthomonas albilineans causing sugarcane leaf scald in China using multilocus sequence analysis

Xanthomonas albilineans is the causal agent of leaf scald, a disease that can cause considerable damage to sugarcane industries. This study analysed the phylogenetic relationship of 14 samples of X. albilineans from China and 13 reference strains retrieved from the GenBank database by multilocus sequence analysis (MLSA). To reach this goal, five housekeeping genes of X. albilineans were amplified from diseased leaves and sequenced: gyrB, abc, rpoD, atpD and glnA. Based on the concatenated sequence of these genes (4473 nt), the 14 samples of X. albilineans from China had 99.9–100% sequence identity with one another and with five strains of the pathogen from the French West Indies and the USA (Florida). The 27 samples or strains of X. albilineans were distributed in two distinct clades in the MLSA‐based phylogenetic tree. Clade 1 was formed by four strains of the pathogen from Fiji, Papua New Guinea and the USA. All the other strains from worldwide locations, including the 14 samples from China, were grouped in clade 2. This latter clade included all strains of the pathogen that were associated with outbreaks of leaf scald that have occurred over the last two decades, especially in the Caribbean islands and the USA. The very low diversity of X. albilineans in four Chinese provinces suggests recent spread of a single strain (from genetic group PFGE‐B) of the leaf scald pathogen within China.

Development of Quantitative Real-Time PCR Assays for Rapid and Sensitive Detection of Two Badnavirus Species in Sugarcane

Sugarcane-infecting badnaviruses (sugarcane bacilliform viruses, SCBVs) represent a genetically heterogeneous species complex, posing a serious threat to the yield and quality of sugarcane in all major producing regions. SCBVs are commonly transmitted across regions by the exchange of sugarcane germplasm. In this study, we develop two quick, sensitive, and reliable protocols for real-time quantitative PCR (qPCR) of Sugarcane bacilliform MO virus (SCBMOV) and Sugarcane bacilliform IM virus (SCBIMV) using two sets of TaqMan probes and primers targeting the reverse transcriptase/ribonuclease H (RT/RNase H) region. The two assays had a detection limit of 100 copies of plasmid DNA and were 100 times more sensitive than conventional PCR. High specificity of the two assays was observed with respect to SCBIMV and SCBMOV. A total of 176 sugarcane leaf tissue samples from Fujian and Yunnan provinces were collected and analyzed in parallel by conventional PCR, SCBIMV-qPCR, and SCBMOV-qPCR. The SCBIMV-qPCR and SCBMOV-qPCR assays indicated that 50% (88/176) and 47% (83/176) samples tested positive, respectively, whereas only 29% (51/176) tested positive with conventional PCR with the primer pairs SCBV-F and SCBV-R. We demonstrate for the first time that SCBIMV and SCBMOV occur in China and reveal coinfection of both Badnavirus species in 29% (51/176) of tested leaf samples. Our findings supply sensitive and reliable qPCR assays for the detection and quantitation of SCBV in sugarcane quarantine programs.