Recovery of bovine lysozyme from transgenic sugarcane stalks: extraction, membrane filtration, and purification.

Increased industrial use of sugarcane (Saccharum spp. hybrid) for food and bioenergy has led to considerable improvements in its genetic transformation, which allowed the development of not only pest- and herbicide-resistant lines but also lines expressing high-value bioproducts and biopolymers. However, the economic benefits of using inexpensive transgenic plant systems for the production of industrial proteins could be offset by high downstream processing costs. In this work, transgenic sugarcane expressing recombinant bovine lysozyme (BvLz) was used to evaluate the feasibility of extraction and fractionation of recombinant proteins expressed in sugarcane stalks. Three pH levels (4.5, 6.0 and 7.5) and three salt concentrations (0, 50, and 150 mM NaCl) were tested to determine BvLz and total protein extractability. Two extraction conditions were selected to prepare BvLz extracts for further processing by cross-flow filtration, a suitable method for concentration and conditioning of extracts for direct applications or prior to chromatography. Partial removal of native proteins was achieved using a 100 kDa membrane but 20-30 % of the extracted BvLz was lost. Concentration of clarified extracts using a 3 kDa membrane resulted in twofold purification and 65 % recovery of BvLz. Loading of concentrated sugarcane extract on hydrophobic interaction chromatography (HIC) resulted in 50 % BvLz purity and 69 % recovery of BvLz.

Detection, Distribution, and Genetic Variability of 'Candidatus Liberibacter' Species Associated with Zebra Complex Disease of Potato in North America.

The specificity and sensitivity of polymerase chain reaction (PCR) primers developed for 'Candidatus Liberibacter solanacearum' and 'Candidatus Liberibacter psyllaurous' were evaluated in conventional and real-time PCR assays. All PCR primers were specific for 'Ca. L. psyllaurous' and 'Ca. L. solanacearum' insomuch as they did not detect other prokaryotic plant pathogens that affect potato except for the putative pathogens associated with psyllid-yellows and haywire. Conventional PCR assays were capable of detecting 0.19 to 1.56 ng of total DNA per reaction, and real-time PCR was found capable of detecting 1.56 to 6.25 ng of total DNA per reaction, depending on the specific PCR primer set used. 'Ca. Liberibacter' species associated with zebra complex disease (ZC) was confirmed in plants affected by this disease throughout Texas from 2005 to 2008, in seed tubers produced in Wyoming in 2007, and in Colorado, Kansas, Nebraska, and Mexico in 2008. A multiplex PCR assay using 'Ca. L. solanacearum'-specific primers and primers specific for the ß-tubulin DNA regions from potato was developed, providing possible utility of the multiplex assay for 'Ca. Liberibacter' detection in different solanaceous plant species. Preliminary studies suggest silverleaf nightshade (Solanum elaeagnifolium), wolfberry (Lycium barbarum), black nightshade (S. ptychanthum), and jalapeno pepper (Capsicum annuum) as additional solanaceous hosts for the ZC-associated bacterium. The 'Ca. Liberibacter' species detected in all samples divided into two clusters sharing similarity of 99.8% in their partial 16S rRNA gene sequences and 99.3% in their partial intergenic spacer region (ISR)-23S rRNA gene sequences. Genetic variation in the 16S rDNA region consistently matched that of the ISR-23S rDNA region. In this partial 16S-ISR-23S rDNA region, there was a total of eight single nucleotide polymorphisms among 'Ca. L. psyllaurous' and 'Ca. L. solanacearum' "strains" investigated in this study. 'Ca. L. solanacearum' and 'Ca. L. psyllaurous' were shown to be very closely related bacteria, if not the same, by successful amplification using a combination of forward primer of 'Ca. L. solanacearum' and reverse primer of 'Ca. L. psyllaurous' in ZC-affected potato samples. This finding clarifies the current taxonomic status of 'Ca. L. solanacearum' and 'Ca. L. psyllaurous'. The detection of 'Ca. L. solanacearum' from haywire-symptomatic potato samples demonstrates that this bacterium might also be associated with this disease.

Mapping the chromosomes of Poncirus trifoliata Raf. by BAC-FISH.

In spite of the importance of Citrus in agriculture and recent progress in genetic mapping and cytogenetics of this group, chromosome mapping of Citrus species is still limited to rDNA probes. In order to obtain a better chromosome characterization of one species from this group, CMA/DAPI double staining followed by in situ hybridization using 45S rDNA and 24 BACs (BAC-FISH) were used on Poncirus trifoliata. The BACs used were obtained from a genomic library of this species and were selected by membrane hybridization using genomic DNA. Four of them were isolated from the Citrus tristeza virus (Ctv) resistance gene region. The P. trifoliata karyotype is composed of two chromosome pairs with one terminal and one proximal CMA(+) band (B type chromosomes), four chromosome pairs with a single CMA(+) band (D type) and three chromosome pairs without bands (F type). In situ hybridization with 13 of the BACs gave single copy signals on seven chromosome pairs. At least one BAC was mapped on each arm of the two B chromosome pairs. Among the four D chromosome pairs, two were identified by BACs mapped on the long arms, one has a 45S rDNA site and the other had no signal. Six BACs allowed identification of the three F chromosome pairs, with one pair hybridizing with four BACs from the Ctv resistance gene region. In summary, all nine chromosome pairs could be differentiated, seven of them by BAC-FISH, while the other two chromosomes could be recognized by the CMA(+) band pattern and 45S rDNA sites. This first BAC-FISH map gives a general framework for comparative genome structure and evolutionary studies in Citrus and Poncirus, allowing the integration of genetic and physical maps when these BACs are included.

Viruses causing mosaic disease in sugarcane and their genetic diversity in southern China.

A survey of cultivated hybrid sugarcane (Saccharum inter-specific hybrid) and noble sugarcane (Saccharum officinarum) in southern China for the presence of Sugarcane mosaic virus (SCMV), Sorghum mosaic virus (SrMV) and Sugarcane streak mosaic virus (SCSMV) was conducted by RT-PCR from the years 2003 to 2006. SCMV and SrMV, but not SCSMV, were found. A high incidence of SCMV and SrMV coinfection was revealed in both hybrid and noble sugarcanes. All coinfected plants showed mosaic symptom, whereas plants infected with a single virus were symptomatic or asymptomatic. It appears that virus mixtures are more virulent than single infections. The nucleotide sequences of the coat protein (CP) gene of 33 SCMV and 10 SrMV isolates from this study were compared to those of CP genes of SCMV and SrMV reported in GenBank. One hundred and seventy-three SCMV isolates, with the exception of MDB and Abaca strains, can be grouped into five groups, which include three previously known groups, the sugarcane (SCE), maize (MZ), and Thailand groups, and two newly identified groups, the noble sugarcane (NSCE) and Brazil groups. Twenty-two SrMV isolates were divided into two groups, HS (hybrid sugarcane) and NS (noble sugarcane) groups. Five out of eight SrMV hybrid isolates belonged to the HS group, and two SrMV noble isolates and three hybrid isolates were within the NS group. Interestingly, the three hybrid isolates within the NS group were isolated from hybrid sugarcane co-infected with SCMV. This indicates that SCMV helps the NS group SrMV to infect hybrid sugarcane.

Citrus tristeza virus transmission by the Toxoptera citricida vector: in vitro acquisition and transmission and infectivity immunoneutralization experiments.

Citrus tristeza virus (CTV) is transmitted by several aphid species in a semi-persistent manner with Toxoptera citricida, the brown citrus aphid (BrCA), being the most efficient. As yet, the molecular interactions between the virus and its aphid vectors have not been determined. This is the first report of aphids acquiring CTV from preparations through an artificial membrane and then transmitting it to receptor plants. The BrCA fed across artificial membranes on crude tissue preparations made from CTV-infected bark tissue were able to transmit CTV to virus-free receptor plants at low rates. CTV p20, p27 and p25 proteins, detected by Western blots, were present in all crude tissue preparations from CTV-infected plants. Partially purified CTV preparations were not transmitted by the BrCA in this manner. Infectivity immunoneutralization experiments were conducted where aphids were forced to feed in vitro on three CTV-specific antibodies (p25, p27 and p20) before being placed on receptor plants following a 48h acquisition feed on CTV-infected source plants. There were no differences in transmission rates among the majority of treatments and the control treatments. However, in one infectivity immunoneutralization experiment, the CTV p20 antibodies significantly enhanced CTV transmission compared to buffer only, pre-immune antiserum or no antibody control treatments. This suggests the inactivity of CTV p20 aids BrCA transmission of virions.

Yellow leaf of sugarcane is caused by at least three different genotypes of sugarcane yellow leaf virus, one of which predominates on the Island of Réunion.

The genetic diversity of sugarcane yellow leaf virus (SCYLV) was analyzed with 43 virus isolates from Réunion Island and 17 isolates from world-wide locations. We attempted to amplify by reverse-transcription polymerase chain reaction (RT-PCR), clone, and sequence four different fragments covering 72% of the genome of these virus isolates. The number of amplified isolates and useful sequence information varied according to each fragment, whereas an amplicon was obtained with diagnostic primers for 59 out of 60 isolates (98%). Phylogenetic analyses of the sequences determined here and additional sequences of 11 other SCYLV isolates available from GenBank showed that SCYLV isolates were distributed in different phylogenetic groups or belonged to single genotypes. The majority of isolates from Réunion Island were grouped in phylogenetic clusters that did not contain any isolates from other origins. The complete six ORFs (5612 bp) of five SCYLV isolates (two from Réunion Island, one from Brazil, one from China, and one from Peru) were amplified, cloned, and sequenced. The existence of at least three distinct genotypes of SCYLV was shown by phylogenetic analysis of the sequences of these isolates and additional published sequences of three SCYLV isolates (GenBank accessions). The biological significance of these genotypes and of the origin of the distinct lineage of SCYLV in Réunion Island remains to be determined.

Severity of Citrus tristeza virus Isolates from Texas.

Citrus tristeza virus (CTV) isolates collected from the Lower Rio Grande Valley in south Texas and east Texas were characterized using citrus indicators and molecular methods. The citrus indicators were Mexican lime (Citrus aurantifolia), sour orange (C. aurantium), sweet orange (C. sinensis) grafted to sour orange, Duncan grapefruit (C. × paradisi), and Madam Vinous sweet orange, with some CTV isolates additionally indexed using the Texas commercial grapefruit cvs. Rio Red and Star Ruby, and Marrs and N-33 sweet orange. Severity ratings used 11 biotype groups or cumulative mean relative indices. Molecular characterization was carried out using poly- and monoclonal antibodies, seven strain-specific probes and single-stranded conformational polymorphism, and all were based on the CTV major coat protein or gene. All Texas CTV isolates produced vein clearing symptoms on inoculated Mexican lime plants. Over half of the CTV isolates tested were placed in biotype groups IX and X (causing decline of sweet orange on sour orange, seedling yellows on sour orange and grapefruit seedlings, and stem pitting of grapefruit or sweet orange), and one isolate was in biotype I (mild).

Effects of snowdrop lectin on Mexican rice borer (Lepidoptera: Pyralidae) life history parameters.

The effects of the snowdrop lectin, Galanthus nivalis agglutinin (GNA), delivered through an artificial diet, on growth, development, and life history parameters of the Mexican rice borer, Eoreuma loftini (Dyar), were evaluated in the laboratory. Incorporation of GNA at three treatment levels, 0.5, 1.0, and 2.0% of total dietary protein, in the larval diet significantly decreased larval survivorship and percentage of adults emerging relative to a control diet lacking GNA, whereas differences were not observed among the three treatment levels. Both larvae and pupae in the control were 8-25% larger than those in the GNA treatments, but differences were not observed between larvae in the GNA treatments. Furthermore, presence of GNA did not affect larval and pupal developmental periods, longevities, and fecundities compared with the control. Mexican rice borer life history parameters, such as net reproductive rate and intrinsic rate of increase, were substantially reduced by the presence of GNA in the diet, but differences were not evident among the three GNA treatment levels.

Evaluation of lectin-expressing transgenic sugarcane against stalkborers (Lepidoptera: Pyralidae): effects on life history parameters.

The impact of snowdrop lectin (Galanthus nivalis agglutinin, GNA) expressed in transgenic sugarcane on life history parameters of Mexican rice borer [Eoreuma loftini (Dyar)] and sugarcane borer [Diatraea saccharalis (F.)] (both Lepidoptera: Pyralidae) was evaluated. In the laboratory, lyophilized sugarcane leaf sheath tissue was incorporated in a meridic diet resulting in a GNA concentration of 0.47% of total protein, and used for insect bioassays over two successive generations. Deleterious effects of GNA were not observed on survival, weight, and developmental periods of larvae and pupae, nor on adult fecundity and egg viability of D. saccharalis. Moreover, in the first generation, addition of transgenic sugarcane tissue to the diet enhanced larval growth in D. saccharalis resulting in higher larval and pupal weight compared with diet with nontransgenic sugarcane, but this effect was not observed in the second generation. In contrast, larval survival, percent adult emergence, and female fecundity of E. loftini were significantly reduced when fed transgenic sugarcane diet compared with nontransgenic sugarcane diet. In addition, a substantial reduction of female pupal weight of E. loftini was observed in the second generation. For both species, the only consistent effect of GNA in both generations was a reduction in adult female longevity. Life table parameters showed that GNA at the level found in the transgenic diet negatively affected development and reproduction of E. loftini, whereas it had a nil to positive effect on development and reproduction of D. saccharalis.

Construction of a 1.2-Mb contig including the citrus tristeza virus resistance gene locus using a bacterial artificial chromosome library of Poncirus trifoliata (L.) Raf.

The citrus tristeza virus resistance gene (Ctv) is a single dominant gene in Poncirus trifoliata, a sexually compatible relative of citrus. To clone this gene, a bacterial artificial chromosome (BAC) library has been constructed from an individual plant that was homozygous for Ctv. This library contains 45,696 clones with an average insert size of 80 kb, corresponding to 9.6 genome equivalents. Screening of the BAC library with five chloroplast DNA probes indicated that 0.58% of the BAC clones contained chloroplast-derived inserts. The chromosome walk across the Ctv locus was initiated using three closely linked genetic markers: C19, AD8, and Z16. The walk has been completed and a contig of ca. 1.2 Mb was constructed. Based on new data, the genetic map in the Ctv region was revised, with Ctv being located between AD8-Z16 and C19 at distances of 1.2 and 0.6 cM, respectively. Utilizing DNA fragments isolated from the contig as RFLP markers, the Ctv locus was further mapped to a region of ca. 300 kb. This contig contains several putative disease-resistance genes similar to the rice Xa21 gene, the tomato Cf-2 gene, and the Arabidopsis thaliana RPS2 gene. This library will therefore allow cloning of Ctv and other putative disease-resistance genes.

Isolation of large terminal sequences of BAC inserts based on double-restriction-enzyme digestion followed by anchored PCR.

Isolation of the terminal portions of genomic DNA cloned in bacterial artificial chromosomes (BACs) is an important step in map-based cloning, and several methods have been developed. Here, we present a new method based on double-restriction-enzyme digestion followed by anchored PCR. BAC DNA was digested with two enzymes: NotI and one of four enzymes (EcoRV, HpaI, StuI, or XmnI) that produce blunt termini. After dephosphorylation, these digestions were ligated to NotI- and EcoRV-digested pMSK, a new cloning vector developed in this work that is derived from pBluescript SK(+). PCR products representing the left- and right-terminal sequences of BAC inserts were obtained using a primer complementary to pMSK and a primer complementary to sequences in either the left arm or the right arm of the BAC vector pBeloBAC11. We have tested this method with 15 different BAC clones, and PCR products representing both the left- and right-terminal sequences have been obtained from all 15 BAC clones. This method is simple, fast, reproducible, and uses the same set of primers for any restriction enzyme used. With some modifications, it can also be used for isolating the terminal portions of genomic DNA cloned in yeast artificial chromosomes and P1-derived artificial chromosomes.

Sugarcane yellow leaf virus: an emerging virus that has evolved by recombination between luteoviral and poleroviral ancestors.

We have derived the genomic nucleotide sequence of an emerging virus, the Sugarcane yellow leaf virus (ScYLV), and shown that it produces one to two subgenomic RNAs. The family Luteoviridae currently includes the Luteovirus, Polerovirus, and Enamovirus genera. With the new ScYLV nucleotide sequence and existing Luteoviridae sequence information, we have utilized new phylogenetic and evolutionary methodologies to identify homologous regions of Luteoviridae genomes, which have statistically significant altered nucleotide substitution ratios and have produced a reconstructed phylogeny of the Luteoviridae. The data indicate that Pea enation mosaic virus-1 (PEMV-1), Soybean dwarf virus (SbDV), and ScYLV exhibit spatial phylogenetic variation (SPV) consistent with recombination events that have occurred between poleroviral and luteoviral ancestors, after the divergence of these two progenitor groups. The reconstructed phylogeny confirms a contention that a continuum in the derived sequence evolution of the Luteoviridae has been established by intrafamilial as well as extrafamilial RNA recombination and expands the database of recombinant Luteoviridae genomes that are currently needed to resolve better defined means for generic discrimination in the Luteoviridae (D'Arcy, C. J. and Mayo, M. 1997. Arch. Virol. 142, 1285-1287). The analyses of the nucleotide substitution ratios from a nucleotide alignment of Luteoviridae genomes substantiates the hypothesis that hot spots for RNA recombination in this virus family are associated with the known sites for the transcription of subgenomic RNAs (Miller et al. 1995. Crit. Rev. Plant Sci. 14, 179-211), and provides new information that might be utilized to better design more effective means to generate transgene-mediated host resistance.

Agrobacterium-mediated transformation of the commercially important grapefruit cultivar Rio Red (Citrus paradisi Macf.).

Transgenic plants of grapefruit cv. Rio Red (Citrus paradisi Macf.) have been obtained by Agrobacterium tumefaciens-mediated gene transfer using seedling-derived epicotyl segments as explants and kanamycin as the selective agent. The transformation procedure includes a shoot elongation phase with a liquid medium overlay, which provides additional selection against non-transgenic shoots. Transformed shoots are invigorated and multiplied on a non-selective medium prior to grafting, thus assuring that plants can be recovered from transgenic shoots. We have constructed a binary vector, pBin34SGUS, with an intron-containing ß-glucuronidase gene (uidA) under the control of the Figwort mosaic virus 34S promoter. The 34S promoter efficiently drives uidA gene expression both in transient assays and in transgenic Rio Red leaf tissue, although at levels five- to sevenfold lower than the Cauliflower mosaic virus 35S promoter. An untranslatable coat protein gene (uncp) of the Citrus tristeza virus strain SY568 and the Galanthus nivalis agglutinin gene (gna) were inserted into pBin34SGUS and transgenic plants have been obtained. Stable integration of the uncp and gna genes was confirmed by Southern hybridization and gna gene expression was confirmed by Western blot analysis.

Molecular characterization of an isolate of citrus tristeza virus that causes severe symptoms in sweet orange.

The complete sequence (19,249 nucleotides) of the genome of citrus tristeza virus (CTV) isolate SY568 was determined. The genome organization is identical to that of the previously determined CTV-T36 and CTV-VT isolates. Sequence comparisons revealed that CTV-SY568, a severe stem-pitting isolate from California, has more than 87% overall sequence identity with CTV-VT, a seedling yellows isolate from Israel. Although SY568 has an overall sequence identity of 81% with CTV-T36, a quick decline isolate from Florida, the sequence identity in the 3' half of the genome is over 90% while the sequence identity in the 5' half of the genome is as low as 56%. Based on the sequence alignments of these three isolates, sequences in the 3' half of the genome are generally well conserved, while the sequences in the 5' half are relatively divergent. Sequence data of independent overlapping clones from the CTV-SY568 genome revealed two regions with highly divergent sequences. In open reading frame 1b (RNA dependent RNA polymerase), there were 118 nucleotide differences that lead to 16 amino acid changes. In the open reading frame of the divergent coat protein gene, 5 amino acid changes result from 48 nucleotide differences. Most differences occurred in the third position of the codons, and resulted in silent amino acid substitutions. RNase protection assays demonstrated that most of the clones obtained are representative of the major RNA species of this isolate. Northern analysis indicated that CTV-SY568 accumulated more viral RNA including genomic and certain subgenomic RNAs than isolates VT or T36 in sweet orange.

Highly sensitive northern hybridization using a rapid protocol for downward alkaline blotting of RNA.

A simple and fast RNA gel blot procedure is described that uses 50 mM NaOH to simultaneously transfer and fix RNA to a positively charged nylon membrane. The RNA is transferred in a downward direction, and transfer is routinely completed within 2.5 h. The resulting blots give increased sensitivity over existing methods without affecting RNA integrity and can be used in both radioactive and nonradioactive detection procedures.

Replication of wild-type and mutant clones of satellite tobacco mosaic virus in Nicotiana benthamiana protoplasts.

RNA transcribed from cloned satellite tobacco mosaic virus (STMV) cDNA replicated in Nicotiana benthamiana protoplasts when co-inoculated with tobacco mild green mosaic virus (TMGMV) genomic RNA, but degraded when inoculated alone. STMV genomic RNA extracted from wild-type virions replicated in protoplasts when co-inoculated with TMGMV, tobacco mosaic virus (TMV) or tomato mosaic virus (ToMV). Transcripts from clones of two STMV coat protein (CP) mutants accumulated to the same level as wild-type transcripts in protoplasts when co-inoculated with TMGMV, whereas a third mutant accumulated to detectable levels in some, but not all, experiments. These results confirm that STMV RNA requires helper virus for replication, and that the helper specificity exhibited by cloned STMV reflects a specific requirement for the TMGMV replicase. It also demonstrates that the low accumulation of STMV CP mutants observed previously in whole plants cannot be attributed to inefficient RNA replication.

Molecular characterization of a bean alpha-amylase inhibitor that inhibits the alpha-amylase of the mexican bean weevil Zabrotes subfasciatus.

Cultivated varieties of the common bean (Phaseolus vulgaris L.) contain an alpha-amylase inhibitor (alpha AI-1) that inhibits porcine pancreatic alpha-amylase (PPA; EC 3.2.1.1) and the amylases of certain seed weevils, but not that of the Mexican bean weevil, Zabrotes subfasciatus. A variant of alpha AI-1, called alpha AI-2, is found in certain arcelin-containing wild accessions of the common bean. The variant alpha AI-2 inhibits Z. subfasciatus alpha-amylase (ZSA), but not PPA. We purified alpha AI-2 and studied its interaction with ZSA. The formation of the alpha AI-2-ZSA complex is time-dependent and occurs maximally at pH 5.0 or below. When a previously isolated cDNA assumed to encode alpha AI-2 was expressed in transgenic tobacco seeds, the seeds contained inhibitory activity toward ZSA but not toward PPA, confirming that the cDNA encodes alpha AI-2. The inhibitors alpha AI-1 and alpha AI-2 share 78% sequence identity at the amino acid level and they differ in an important region that is part of the site where the enzyme binds the inhibitor. The swap of a tripeptide in this region was not sufficient to change the specificity of the two inhibitors towards their respective enzymes. The three-dimensional structure of the alpha AI-1/PPA complex has just been solved and we recently obtained the derived amino acid sequence of ZSA. This additional information allows us to discuss the results described here in the framework of the amino acid residues of both proteins involved in the formation of the enzyme-inhibitor complex and to pinpoint the amino acids responsible for the specificity of the interaction.

Outbreak of Sugarcane Mosaic in Commercial Sugarcane Cultivar CP 72-2086, in Florida.

In May 1996, an outbreak of sugarcane mosaic virus (SCMV) was detected in Florida affecting a major commercial sugarcane (Saccharum spp. hybrid) cultivar, CP 72-2086. Identification of SCMV was confirmed by enzyme-linked immunosorbent assay (ELISA) with SCMV antiserum (ATCC no. PVAS 115), and by reverse transcription(RT)-PCR (1,2). The strain of SCMV was identified as E with an RT-PCR-based restriction fragment length polymorphism analysis (1). Although SCMV strain E has been detected in Florida for many decades, it had been confined primarily to S. officinarum clones, to occasional susceptible sugarcane clones in breeding programs, and to various grasses throughout the sugarcane production area. CP 72-2086 plus seven other sugarcane cultivars and 10 sorghum cultivars reacted similarly to inoculation with the isolate of SCMV found recently on CP 72-2086 and an isolate of SCMV collected in 1986. CP 72-2086 was released in 1982 and currently is the second most widely grown cultivar, constituting 18.0% of the commercial sugarcane area in Florida. At the epicenter of infection, located 11 km southeast of Canal Point, over 50% of CP 72-2086 plants had SCMV symptoms. The incidence of mosaic decreased rapidly away from the epicenter. No SCMV was observed in the western area of the sugarcane production area, west of South Bay, FL. References: (1) T. E. Mirkov and J. E. Irvine. Sugar y Azucar 9:23, 1996. (2) G. R. Smith et al. Plant Dis. 78:557, 1994.

Sequence and Relationships of Sugarcane Mosaic and Sorghum Mosaic Virus Strains and Development of RT-PCR-Based RFLPs for Strain Discrimination.

ABSTRACT Sugarcane mosaic is the most widespread virus disease affecting sugarcane production. We have established a collection of seven prominent sugarcane mosaic potyvirus (SCMV) strains currently causing disease in sugarcane throughout the world and originally found in sugarcane in the United States. This collection includes SCMV strains A, B, D, and E, and the sorghum mosaic virus (SrMV) strains SCH, SCI, and SCM. These viruses were propagated on Sorghum bicolor cv. Rio and purified. Cloned cDNAs representing 2.0 kb of the 3' termini, obtained after a reversetranscriptase-polymerase chain reaction (RT-PCR) on purified virions using an oligo(dT) primer and degenerate primers with sequences located in the NIb gene, have been sequenced for each of these strains. A comparative analysis of the deduced amino acid sequences in the NIb and coat protein genes and of the nucleotide sequences in the 3'-untranslated region, among these seven viruses and among six other members of the SCMV subgroup, confirms that there are at least four, but suggests that there are five, distinct viruses in the SCMV complex. Based on these seven new sequences and on the available sequence data for six other members of the SCMV subgroup, we have developed group-specific primers for use in RT-PCR-based restriction fragment length polymorphism analysis for rapid discrimination between strains of SCMV and SrMV. This is the first assay for differentiating strains of SCMV and SrMV that does not require interpretation of symptoms on differential hosts.

A putative precursor protein in the evolution of the bean alpha-amylase inhibitor.

Seeds of the common bean Phaseolus vulgaris and the tepary bean (P. acutifolius) contain a family of plant defence proteins that includes phytohaemagglutinin (PHA), arcelin and alpha-amylase inhibitor (alpha AI). These homologous proteins differ by the absence of short loops at the surface of the protein and by the presence of a proteolytic processing site (Asn77) that allows alpha AI to be post-translationally cleaved and activated. We now report the derived amino acid sequence of two amylase inhibitor-like (AIL) proteins that are not proteolytically processed, although they have the typical processing site. One protein is from the common bean, and the other from the tepary bean. On a dendrogram, these proteins are grouped with alpha AIs rather than with the arcelins or lectins. alpha AI differs from AIL primarily by the deletion of a 15-amino-acid segment from the middle of the AIL sequence. When alpha AI is expressed in tobacco, it is proteolytically processed to form an active molecule. However, AIL sequences are not processed. We suggest that the AIL proteins may be an intermediate in the evolution of an active alpha AI.