BACKGROUND
the gibberellin stimulated-like proteins gsl <dig>  and gsl <dig>  are cysteine-rich peptides from potato  with in vitro antimicrobial activity against a wide range of bacteria and fungi  <cit> , as well as nematodes  <cit> .
the spectrum of antimicrobial activity is almost identical for gsl <dig> and gsl <dig>  <cit> .
gsl <dig> and gsl <dig> induce rapid aggregation of both gram-negative and gram-positive bacteria, and although this response does not correlate with antimicrobial activity, it is still considered that these proteins may play an in vivo role in controlling pathogen migration  <cit> .
amino acid sequence alignment of gsl <dig> and gsl <dig> show similarity with the gast <dig>  from tomato  <cit>  and the gasa family  from arabidopsis  <cit>  and similar members from a wide range of dicotyledonous and monocotyledonous species  <cit> .
based on a limited similarity in amino acid sequence to the hemotoxic, desintegrin-like snake venoms, gsl <dig> and gsl <dig> were formerly referred to as snakin- <dig> and snakin- <dig>  <cit> .
however, the term snakin is inappropriate for these plant-based proteins since gsl <dig> and gsl <dig> do not share the rgd residues and functional properties of snake venoms responsible for desintegrin action  <cit> .
both the stgsl <dig> and stgsl <dig> genes encode polypeptides that have similar structural features with an n-terminal putative signal sequence congruent with a sub-cellular location in the plant cell wall and a cysteine-rich c-terminal domain  <cit> .
gsl <dig> has a signal sequence of  <dig> amino acid residues, followed by a 63-amino acid mature peptide  with  <dig> highly conserved cysteine residues  <cit> .
gsl <dig> has a  <dig> amino acid-residue signal peptide, followed by an intermediate 15-residue acidic peptide, and then a mature peptide  of a  <dig> amino acid basic peptide with the  <dig> conserved cysteine residues  <cit> .
gsl <dig> and gsl <dig> peptides share several features characteristic of all antimicrobial peptides.
the cysteine-rich nature of these peptides is critical for the occurrence of disulphide bridges that are important for enhancing the structural stability under diverse stress conditions  <cit> .
a high frequency of charged amino acids appears to play a key role in the activity against microbes  <cit> , along with the amphipathic structure and cationic charge at physiological ph  <cit> .
the prediction of gsl <dig> three-dimensional structure and disulfide bonding pattern revealed two long α-helices stabilized and maintained by six knotted disulfide bonds between specific cysteine residues  <cit> .
northern analysis in potatoes established that transcripts of stgsl <dig> exhibited highest accumulation in stems, shoot apices, young floral buds and petals, with expression also detected in tubers and carpels, but not in roots, stolons, leaves, sepals or stamens  <cit> .
transcripts in leaves were not induced by either abiotic or biotic stress, or chemical treatments such as jasmonic acid, salicylic acid, isonicotinic acid, abscisic acid, gibberellic acid, and indolacetic acid, leading to the conclusion that gsl <dig> is a component of the constitutive defence barriers, especially of the storage and reproductive organs  <cit> .
similar studies on stgsl <dig> expression detected the highest accumulation of transcripts in tubers, petals and carpels, with expression also in stems, shoot apices, leaves, flower buds and stamens, but not in roots, stolons and sepals  <cit> .
in contrast to stgsl <dig>  the stgsl <dig> gene was locally up-regulated in leaves by wounding and abscisic acid treatments, responded weakly to salinity stress, while drought stress or treatments with gibberellic acid, chitosan, jasmonic acid, ethylene, benzothiadiazole-7-carbothioic acid or s-methyl ester had no effect  <cit> .
stgsl <dig> expression was also up-regulated upon infection of tubers with the compatible fungus botrytis cinerea, but down-regulated by the bacterial pathogens ralstonia solanacearum and dickeya chrysanthemi , resulting in the overall hypothesis that gsl <dig> is a component of both constitutive and inducible defence barriers to pathogens  <cit> .
over-expression of the stgsl <dig> gene in transgenic potato plants enhances resistance against two important potato pathogens pectobacterium carotovorum subspecies carotovorum  and rhizoctonia solani <cit> .
transgenic wheat plants over-expressing the solanum chacoense gsl <dig> gene exhibited improved resistance to blumeria graminis f.sp.
tritici <cit> .
likewise, over-expression of the tomato gsl <dig> gene in tomato enhanced tolerance to clavibacter michiganensis subsp.
michiganensis that causes bacterial canker and wilt disease  <cit> .
viral-induced gene silencing of gsl <dig> in nicotiana benthamiana increased susceptibility to wilt disease development induced by c. michiganensis subsp.
michiganensis <cit> .
similarly, virus-induced gene silencing of gsl <dig> in capsicum annuum increased susceptibility to root-knot nematodes   <cit> .
a defence role for gsl <dig> was also suggested from the observation of decreased virulence of gsl1-sensitive mutants of dickeya chrysanthemi  to potato tubers  <cit> .
the antimicrobial mechanism of action for gsl peptides is not known, but in contrast to other antimicrobial peptides from plants, gsl <dig> and gsl <dig> do not interact with artificial lipid membranes  <cit> .
a cysteine-rich  <dig>  kda orthologue of gsl <dig> from french bean  was demonstrated to tightly bind to a 25 kda polypeptide of a proline-rich protein family from legumes and thought to function as a two-component chitin-receptor involved in plant-pathogen interactions through antimicrobial activity and/or signalling  <cit> .
there is no consensus on the biological roles of gsl proteins.
given their in vitro antimicrobial activity they are often considered to play important roles in the innate defence against invading microorganisms  <cit>  and/or to be a key determinant during the interaction between plants and pathogens  <cit> .
similar genes in other species have been implicated in diverse biological processes, including: cell division, cell elongation, cell growth, transition to flowering, somatic embryogenesis and signalling pathways  <cit> .
despite the highly conserved nature of gsl/gasa amino acid sequences, including  <dig> cysteine residues at the c-terminus that are probably responsible for the protein structure and biochemical activity, the functions and mode of action of gsl/gasa proteins are not completely elucidated  <cit> .
the prevailing view is that gsl/gasa proteins play a role in several aspects of plant development, plant responses to biotic or abiotic stress through their participation in hormone crosstalk, and redox homeostasis  <cit> .
this is supported by partial silencing of gsl <dig> in potato using antisense approaches that resulted in plants with reduced height and smaller leaves resulting from reduced cell division, altered leaf metabolism and cell wall composition  <cit> .
to help resolve the biological roles of gsl <dig> and gsl <dig> peptides we have undertaken a thorough analysis of the structure and expression of these genes in potato.
we have characterised the full length genes for both gsl <dig> and gsl <dig> from diploid and tetraploid potato using the genome sequence of potato  <cit> , coupled with further next generation sequencing of highly heterozygous tetraploid cultivars.
specific promoter motifs and exon regions are highly conserved among multiple alleles, suggesting their importance for biological function.
analysis of comprehensive transcriptome data substantiates the role of specific promoter motifs in transcriptional control of gene expression.
the lethality of antisense knock-down expression suggests the essential role of this gene family in potatoes.
RESULTS
allelic polymorphism of gsl genes in potato
pcr isolation, cloning and sequencing of the coding region from the autotetraploid potato cultivar ‘iwa’ revealed two alleles  for the gsl <dig> gene  and two alleles  for the gsl <dig> gene .
from the frequency of clones with the gsl <dig> alleles, it is estimated that iwa has three copies of the a <dig> allele  and one copy of the a <dig> allele .
similarly for the gsl <dig> gene, iwa has three copies of the b <dig> allele  and one copy of the b <dig> allele .
the a <dig> and a <dig> alleles of the gsl <dig> gene differed by  <dig> single nucleotide polymorphisms  and four indels of 1– <dig> nucleotides.
all of these variant nucleotides were in the introns, except for only three synonymous snps in the exons.
similarly, the b <dig> and b <dig> alleles of the gsl <dig> gene were polymorphic for  <dig> snps and five indels of 1– <dig> nucleotides, with only four synonymous snps all occurring in the third exon of gsl <dig>
pcr isolation and direct sequencing of cdna products determined the sequence of cdna from mature transcripts for both the gsl <dig>  and gsl <dig>  genes.
alignment of the genomic and cdna sequences confirmed the exon and intron regions in both the gsl <dig> and gsl <dig> genes .
the gsl <dig> gene consists of two exons of  <dig> and  <dig> nucleotides interrupted by a single intron of  <dig> nucleotides.
the gsl <dig> gene is composed of three exons of  <dig>   <dig> and  <dig> nucleotides respectively, interspersed with two introns of  <dig> and  <dig> nucleotides.
the exon/intron boundaries were identical in all alleles of both the gsl <dig> and gsl <dig> genes, with the splice sites possessing the conserved 5’gt and 3’ag dinucleotides , consistent with the consensus sequences of the intron at both donor and acceptor sites  <cit> .
structure of full length gsl genes
the sequence of the coding regions of the gsl <dig> and gsl <dig> genes described above were used for interrogation of the potato genome sequence  <cit> .
firstly, gsl <dig> and gsl <dig> nucleotide sequences were used to blast search the reference cds and genomic sequences to identify gene locations on superscaffold assemblies.
secondly, annotated protein sequences from potato genome assembly version  <dig>  of the genotype dm1- <dig>  <dig> r <dig>  were also searched for the presence of the gasa pfam motif pf <dig> .
results showed single copy genes for each of gsl <dig> and gsl <dig>  gsl <dig> is located on superscaffold pgsc0003dmb <dig> which has been mapped to chromosome  <dig>  whereas gsl <dig> is located on superscaffold pgsc0003dmb <dig> which has been mapped to chromosome  <dig> .
several other genes similar to gsl and gasa genes were also identified; results of these searches are shown in additional file 3: table s <dig>
the general structure of the gsl <dig> and gsl <dig> genes was analysed based on the dm sequence of the potato genome  <cit> .
for the gsl <dig> gene, up to  <dig> nucleotides were recovered upstream from the putative transcription start site.
we were only able to confirm up to  <dig> nucleotides upstream of the putative transcription start site for the gsl <dig> gene due to a gap in the assembly of the reference potato genome in the upstream region of the gsl <dig> promoter.
however, we documented at least  <dig> nucleotides downstream of the stop codon for both gsl genes.
a putative transcription start site was predicted based on a plant dimer motif yr rule  <cit>  at  <dig> nucleotides and  <dig> nucleotides upstream from the first base of the translation start site  for the gsl <dig> and gsl <dig> genes, respectively.
putative cis-elements were identified for the gsl <dig> gene .
these are tata-box , a pyrimidine patch  and caat-box .
in gsl <dig>  a putative tata-box and y patch were located from nucleotides − <dig> to − <dig> and nucleotides − <dig> to − <dig>  respectively.
since we were unable to locate a satisfactory caat-box, we identified a ‘hypothetical’ caat-box  in the gsl <dig> promoter .
next generation sequencing and snp discovery
genomic regions for gsl <dig> and gsl <dig> were further analyzed for sequence variation by aligning re-sequence data generated from illumina reads of the diploid rh89-039- <dig>   <cit>  and data generated from four tetraploid lines using the illumina gaiix platform.
illumina short insert read pair data were generated for each line.
reads were aligned to the reference genome using bwa  <cit> .
alignments were further analysed using samtools  <cit> , polysnp  and visualized using geneious  <cit> .
for the diploid rh and the four tetraploid potato genotypes the structure of the gsl <dig> and gsl <dig> genes and sequence polymorphisms for the various alleles were annotated manually and compared to the reference dm potato genome.
the locations of snps and indels  identified across all alleles from all genotypes is illustrated in figure  <dig> and summarized in table  <dig>  in gsl <dig> multiple snps and indels were identified within the non-coding regions, with the greatest frequency occurring within the intron and the region from − <dig> to − <dig> nucleotide positions relative to the 5’utr.
the 5’utr contains only a single snp that is found in one re-sequenced genotype.
the exon regions have no indels and only very rare synonymous snps, with an overall snp frequency in exons of one snp/ <dig> nucleotides .
in contrast, the single intron contains  <dig> snps and  <dig> different indels.
the gsl <dig> gene also exhibits the highest snp frequency within the introns .
a deletion of  <dig> nucleotides was found within the 5’utr of dm, but not in the other genotypes.
other indels were found in all gene components except the exons.
consistent with the gsl <dig> gene, the exons of gsl <dig> exhibit only very rare synonymous snps among the alleles from all genotypes, with an overall snp frequency in exons of one snp/ <dig> nucleotides .
gsl <dig>
and
gsl <dig>
genes
the next generation sequence data from four tetraploid potato genotypes , plus the diploid rh  <cit> , were aligned with the reference potato genome of dm  <cit> .
snp output is from polysnp tool with stringent calling .
the rare frequency of snps observed in gsl <dig> and gsl <dig> within and between the diploid homozygous dm, the diploid heterozygous rh, and the four tetraploid potato genotypes was comparable to other gsl and gasa-like genes, as well as other highly conserved housekeeping genes .
the rare snp incidence in these genes across these  <dig> haplotypes is substantially lower than the one snp every  <dig> nucleotides observed in a  <dig>  mb region analyzed for only two rh haplotypes associated with the potato genome sequence  <cit> , and the one snp every  <dig> nucleotides  and one snp every  <dig> nucleotides  reported by uitdewilligen et al.
<cit>  based on targeted resequencing of  <dig> tetraploid cultivars.
this confirms the highly conserved nature of the gsl <dig> and gsl <dig> genes, and therefore suggests that they play an essential role in biological function.
expression profiles of gsl genes
fpkm  as expression values for each transcript were extracted from previous data sets  <cit>  representing a range of different potato tissues and treatments .
fpkm levels from a total of  <dig> tissue and treatment libraries from the genotype dm were analyzed for gsl <dig> and gsl <dig> expression.
of the  <dig> samples analyzed, gsl <dig> was expressed at a higher level in  <dig> samples compared with gsl <dig>  often by over an order of magnitude in fpkm values.
for gsl <dig> expression, segura et al.
<cit>  showed by northern analysis that transcripts of stgsl <dig> exhibited highest accumulation in stems, shoot apices, young floral buds and petals.
similarly, fpkm reads showed highest levels in mature flowers, immature fruit, shoots, petals and carpels .
northern analysis also detected expression in tubers and carpels, but not in roots, stolons, leaves, sepals or stamens  <cit> .
in contrast to the northern analysis, fpkm analysis showed that gsl <dig> was expressed in roots, stolons, leaves and sepals.
northern analysis and fpkm data are in agreement for the absence of gsl <dig> expression in stamens; with the fpkm data also showing no expression in mature fruit and callus tissue.
northern analysis also established gsl <dig> expression is not induced by biotic or abiotic stresses  <cit> , while the fpkm data show an absence of gsl <dig> transcripts in response to heat stress or baba treatments and a slight increase in transcripts in response to bth treatment .
analysis of arabidopsis thaliana plants transgenic for gus fusions to the potato gsl <dig> promoter revealed gus expression in root vascular tissue, cotyledons, young leaves and floral organs  <cit> .
this analysis of transcriptional control by the gsl <dig> promoter is more consistent with the fpkm data  than the previously published northern analysis  <cit> .
fpkm values for the gsl <dig> gene  and northern analysis  <cit>  are in agreement with highest levels of expression being in carpels and petals, and generally high expression in all tissues examined.
however, northern analysis did not detect gsl <dig> expression in stolons or roots, which is in contrast to the high levels of expression seen in fpkm data .
in addition, fpkm analysis indicates that gsl <dig> expression is not induced by biotic or abiotic stresses, plant growth regulator treatments or wounding , although slight reductions are observed in gsl <dig> transcripts in response to stress induced by heat, salt, mannitol and baba treatments.
this is in contrast with northern analysis, where gsl <dig> expression responded to biotic stress, was up-regulated by wounding and aba treatments, down regulated in response to ga <dig> and showed no response to salinity or drought treatments  <cit> .
the differences between previously published northern analysis for gsl <dig> <cit>  and gsl <dig> <cit>  and the fpkm values in this study  may reflect differences in cultivar/ploidy level and growth/treatment conditions.
overall, the fpkm data support the conclusion that gsl <dig> is a component of the constitutive defense barriers, especially of the storage and reproductive organs  <cit> .
the fpkm analysis supports the same conclusion for gsl <dig>  which is in contrast to the previous view that gsl <dig> is a component of both constitutive and inducible defense barriers  <cit> .
analysis of gsl promoters
since the gsl <dig> and gsl <dig> genes differed markedly in their magnitude and specificity of transcript accumulation , the promoter regions were analysed for motifs using genomatix-matinspector  <cit>  based on place  <cit> .
a total of  <dig> and  <dig> different motifs, previously characterised in other studies, were identified in the gsl <dig> and gsl <dig> promoter regions, respectively .
based on the known transcriptional expression of the gsl genes , the putative roles of gsl proteins  <cit> , the repeated occurrence of motifs, their presence in all potato genotypes, and their relative position in the promoter region, key motifs with potential functional significance were identified for gsl <dig>  and gsl <dig> .
gsl <dig>
promoter region
analysis used genomatix-matinspector  <cit>  based on place  <cit> .
the approximate positions of the motif numbers are indicated on figure 1a.
aall four tetraploid genotypes contained all motifs.
however, allelic polymorphisms involving snps were occasionally observed in some motifs of these genotypes as indicated by c , k , s , and v .
#indicates an indel in the motif of at least one allele of the indicated genotype.
gsl <dig>
promoter region
analysis used genomatix-matinspector  <cit>  based on place  <cit> .
the approximate positions of the motif numbers are indicated on figure 1b.
aall four tetraploid genotypes contained all motifs.
however, allelic polymorphisms involving snps were occasionally observed in some motifs of these genotypes as indicated by c , k , s , and v .
v# indicates an insertion into the motif in at least one allele of vtn62-33- <dig>
eight different key motifs were identified in the gsl <dig> promoter, which are repeated up to eleven times resulting in a total of  <dig> motifs .
these involve motifs associated with roles for response to disease and biotic stress, abiotic stresses, light induction, and plant development and were found in the genome sequence of dm as well as all four tetraploid genotypes.
however, allelic polymorphisms involving disruptions of these motifs were occasionally observed in the tetraploid genotypes.
polymorphic snps were observed in seven of the  <dig> motifs, with polymorphic indels also observed for nine motifs .
in the gsl <dig> promoter one key motif was identified that occurs five times and ten other key motifs were identified that occur only once .
similar to the promoter of the gsl <dig> gene, these have known roles associated with biotic stress, abiotic stress, and development.
additional motifs present in the gsl <dig> promoter are associated with sugar signaling and hormone responses.
these fifteen motifs were all observed in the four tetraploid genotypes, although two were observed to be polymorphic for snps and one was polymorphic for an insertion into at least one allele of the genotype vtn62-33- <dig> .
the conservation of these motifs across the genome of dm and all four re-sequenced tetraploid genotypes substantiates their importance.
their presence in the gsl <dig> and gsl <dig> promoter regions aligns with the transcriptional expression of the respective genes observed by previous northern analysis  <cit>  and/or the fpkm data .
the presence of allelic polymorphisms involving sequence disruptions in some of these motifs could be representative of alleles with potentially altered transcriptional expression of the gsl <dig> and gsl <dig> genes.
antisense knockdown of gsl expression
using our standard agrobacterium-mediated transformation protocol for potato  <cit> , we failed to recover any transformants of potato cultivar iwa with antisense constructs for either the gsl <dig> or gsl <dig> genes.
over  <dig> leaf explants were subjected to agrobacterium-mediated transformation in each of three experiments for both gsl genes using our well established protocol.
we would normally expect to recover at least one regenerated transformant per leaf explant for the potato cultivar iwa when selecting for the kanamycin resistance marker gene used on the binary vector.
this expected frequency was achieved in concurrent related experiments using the gsl sense constructs  <cit> .
however, a total of only  <dig> and  <dig> putative transformed cell colonies were recovered from all three co-cultivation experiments with agrobacterium containing the gsl <dig> and gsl <dig> antisense constructs, respectively .
all potato cell colonies transformed with the antisense constructs failed to continue growth and eventually senesced and died before complete shoots were regenerated .
the senescing cultures were sub-cultured onto medium without timentin™ and dna was extracted from those exhibiting no agrobacterium growth.
pcr using primers that bridged the lhca <dig> promoter and the antisense gsl coding regions confirmed that these cell colonies were transformed with the intended construct prior to their death .
the same dna samples failed to amplify pcr products using primers specific to the agrobacterium virg gene.
this confirms the absence of agrobacterium cells in the plant tissue that would otherwise compromise the pcr testing of the transformed potato cell colonies.
the lethality of antisense knock-down expression of gsl <dig> and gsl <dig> suggests an essential role of the gsl gene family for potato development.
a previous study achieved partial silencing of gsl <dig> in potato by expressing an antisense rna under the control of the 35s promoter.
this resulted in plants with reduced height and smaller leaves resulting from reduced cell division, changed leaf metabolism and cell wall composition  <cit> .
the lhca <dig> st. <dig> promoter used in the present study is known to confer higher and more stable transgene expression than the 35s promoter  <cit> .
consequently, the lethality of gsl <dig> and gsl <dig> antisense knock-down under the control of the lhca <dig> promoter is not unexpected given the dramatic phenotypes observed with the partial silencing from the use of the 35s promoter  <cit> .
it is plausible that these gsl <dig> or gsl <dig> knock-down impacts, resulting from antisense expression driven by either the 35s or the lhca <dig> promoters, could also arise by interference in expression of other closely related gsl and gasa genes.
the three most closely related genes to gsl <dig> show 68-78% identity in exon regions, whereas the identity with all the other related genes was only 42-56% .
for gsl <dig>  the related gsl and gasa genes have only 44-60% identity in exon regions .
although this level of identity may be sufficient to trigger a partial knock-down of these related genes, it is unlikely to result in complete knock-down necessary for lethality.
therefore, the lethality of gsl <dig> and gsl <dig> antisense expression under the control of the lhca <dig> promoter can be attributed to knock-down of the gsl <dig> and gsl <dig> genes.
CONCLUSIONS
gsl <dig> and gsl <dig>  gibberellin stimulated-like proteins , are cysteine-rich peptides from potato  with antimicrobial properties  <cit> .
given their in vitro antimicrobial activity, the gsl <dig> and gsl <dig> genes are often considered to play important roles in the innate defence against invading microorganisms  <cit>  and/or to be a key determinant during the interaction between plants and pathogens  <cit> .
in other species similar gsl/gasa proteins are hypothesised to play diverse biological roles in several aspects of plant development, plant responses to biotic or abiotic stress through their participation in hormone crosstalk, and redox homeostasis  <cit> .
to further the understanding of the biological roles of gsl proteins, we undertook a thorough analysis of the structure and expression of these genes in potato.
we isolated and sequenced the coding regions and cdnas for both gsl <dig> and gsl <dig> genes from the potato cultivar iwa.
this revealed two alleles  for the gsl <dig> gene  and two alleles  for the gsl <dig> gene .
alignment of the genomic and cdna sequences confirmed the exon and intron regions in both the gsl <dig> and gsl <dig> genes .
the gsl <dig> gene consists of two exons of  <dig> and  <dig> nucleotides interrupted by a single intron of  <dig> nucleotides.
the gsl <dig> gene is composed of three exons of  <dig>   <dig> and  <dig> nucleotides respectively, alternating with two introns of  <dig> and  <dig> nucleotides.
we have also characterised the full length genes for both gsl <dig>  and gsl <dig>  using the genome sequence of diploid potato  <cit> , coupled with further next generation sequencing of four highly heterozygous tetraploid potato genotypes; cultivars summer delight and karaka, and breeding lines 1021/ <dig> and vtn62-33- <dig>  the frequency of snps in gsl <dig> and gsl <dig> was very low with only one snp every  <dig> and  <dig> nucleotides in exon regions of gsl <dig> and gsl <dig>  respectively , similar to other highly conserved housekeeping genes in potato .
specific promoter motifs were also highly conserved among multiple alleles representing the  <dig> haplotypes from dm and the four re-sequenced tetraploid genotypes , suggesting their importance for biological function.
analysis of comprehensive rna-seq data substantiated the role of specific promoter motifs in transcriptional control of gene expression .
fpkm analysis established that gsl <dig> was expressed at a higher level than gsl <dig> in  <dig> out of  <dig> libraries, often by an order of magnitude.
furthermore, both the gsl <dig> and gsl <dig> genes exhibited constitutive expression that was not up-regulated in response to biotic or abiotic stresses, hormone treatments or wounding.
the fpkm analysis did not always agree with previous northern analysis  <cit> , although closely matched conclusions from the analysis of arabidopsis thaliana plants transgenic for gus fusions to the potato gsl <dig> promoter  <cit> .
the gsl <dig> and gsl <dig> genes from potato are very highly conserved suggesting they contribute to an important biological function.
the known antimicrobial activity of the gsl proteins, coupled with the fpkm analysis from rna-seq data, suggests that both genes contribute to the constitutive defence barriers in potatoes.
the lethality of antisense knock-down expression of gsl <dig> and gsl <dig>  coupled with the rare incidence of snps in these genes, suggests an essential role for this gene family.
these features are consistent with the gsl protein family playing a role in several aspects of plant development and plant defence responses.

 the gibberellin stimulated-like proteins gsl and gsl are cysteine-rich peptides from potato with in vitro antimicrobial activity against a wide range of bacteria and fungi, as well as nematodes. the spectrum of antimicrobial activity is almost identical for gsl and gsl. gsl and gsl induce rapid aggregation of both gram-negative and gram-positive bacteria, and although this response does not correlate with antimicrobial activity, it is still considered that these proteins may play an in vivo role in controlling pathogen migration. amino acid sequence alignment of gsl and gsl show similarity with the gast from tomato and the gasa family from arabidopsis and similar members from a wide range of dicotyledonous and monocotyledonous species. based on a limited similarity in amino acid sequence to the hemotoxic, desintegrin-like snake venoms, gsl and gsl were formerly referred to as snakin- and snakin-. however, the term snakin is inappropriate for these plant-based proteins since gsl and gsl do not share the rgd residues and functional properties of snake venoms responsible for desintegrin action. both the stgsl and stgsl genes encode polypeptides that have similar structural features with an n-terminal putative signal sequence congruent with a sub-cellular location in the plant cell wall and a cysteine-rich c-terminal domain. gsl has a signal sequence of amino acid residues, followed by a 63-amino acid mature peptide with highly conserved cysteine residues. gsl has a amino acid-residue signal peptide, followed by an intermediate 15-residue acidic peptide, and then a mature peptide of a amino acid basic peptide with the conserved cysteine residues. gsl and gsl peptides share several features characteristic of all antimicrobial peptides. the cysteine-rich nature of these peptides is critical for the occurrence of disulphide bridges that are important for enhancing the structural stability under diverse stress conditions. a high frequency of charged amino acids appears to play a key role in the activity against microbes, along with the amphipathic structure and cationic charge at physiological ph. the prediction of gsl three-dimensional structure and disulfide bonding pattern revealed two long α-helices stabilized and maintained by six knotted disulfide bonds between specific cysteine residues. northern analysis in potatoes established that transcripts of stgsl exhibited highest accumulation in stems, shoot apices, young floral buds and petals, with expression also detected in tubers and carpels, but not in roots, stolons, leaves, sepals or stamens. transcripts in leaves were not induced by either abiotic or biotic stress, or chemical treatments such as jasmonic acid, salicylic acid, isonicotinic acid, abscisic acid, gibberellic acid, and indolacetic acid, leading to the conclusion that gsl is a component of the constitutive defence barriers, especially of the storage and reproductive organs. similar studies on stgsl expression detected the highest accumulation of transcripts in tubers, petals and carpels, with expression also in stems, shoot apices, leaves, flower buds and stamens, but not in roots, stolons and sepals. in contrast to stgsl the stgsl gene was locally up-regulated in leaves by wounding and abscisic acid treatments, responded weakly to salinity stress, while drought stress or treatments with gibberellic acid, chitosan, jasmonic acid, ethylene, benzothiadiazole-7-carbothioic acid or s-methyl ester had no effect. stgsl expression was also up-regulated upon infection of tubers with the compatible fungus botrytis cinerea, but down-regulated by the bacterial pathogens ralstonia solanacearum and dickeya chrysanthemi, resulting in the overall hypothesis that gsl is a component of both constitutive and inducible defence barriers to pathogens. over-expression of the stgsl gene in transgenic potato plants enhances resistance against two important potato pathogens pectobacterium carotovorum subspecies carotovorum and rhizoctonia solani. transgenic wheat plants over-expressing the solanum chacoense gsl gene exhibited improved resistance to blumeria graminis f.sp. tritici. likewise, over-expression of the tomato gsl gene in tomato enhanced tolerance to clavibacter michiganensis subsp. michiganensis that causes bacterial canker and wilt disease. viral-induced gene silencing of gsl in nicotiana benthamiana increased susceptibility to wilt disease development induced by c. michiganensis subsp. michiganensis. similarly, virus-induced gene silencing of gsl in capsicum annuum increased susceptibility to root-knot nematodes. a defence role for gsl was also suggested from the observation of decreased virulence of gsl1-sensitive mutants of dickeya chrysanthemi to potato tubers. the antimicrobial mechanism of action for gsl peptides is not known, but in contrast to other antimicrobial peptides from plants, gsl and gsl do not interact with artificial lipid membranes. a cysteine-rich  kda orthologue of gsl from french bean was demonstrated to tightly bind to a 25 kda polypeptide of a proline-rich protein family from legumes and thought to function as a two-component chitin-receptor involved in plant-pathogen interactions through antimicrobial activity and/or signalling. there is no consensus on the biological roles of gsl proteins. given their in vitro antimicrobial activity they are often considered to play important roles in the innate defence against invading microorganisms and/or to be a key determinant during the interaction between plants and pathogens. similar genes in other species have been implicated in diverse biological processes, including: cell division, cell elongation, cell growth, transition to flowering, somatic embryogenesis and signalling pathways. despite the highly conserved nature of gsl/gasa amino acid sequences, including cysteine residues at the c-terminus that are probably responsible for the protein structure and biochemical activity, the functions and mode of action of gsl/gasa proteins are not completely elucidated. the prevailing view is that gsl/gasa proteins play a role in several aspects of plant development, plant responses to biotic or abiotic stress through their participation in hormone crosstalk, and redox homeostasis. this is supported by partial silencing of gsl in potato using antisense approaches that resulted in plants with reduced height and smaller leaves resulting from reduced cell division, altered leaf metabolism and cell wall composition. to help resolve the biological roles of gsl and gsl peptides we have undertaken a thorough analysis of the structure and expression of these genes in potato. we have characterised the full length genes for both gsl and gsl from diploid and tetraploid potato using the genome sequence of potato, coupled with further next generation sequencing of highly heterozygous tetraploid cultivars. specific promoter motifs and exon regions are highly conserved among multiple alleles, suggesting their importance for biological function. analysis of comprehensive transcriptome data substantiates the role of specific promoter motifs in transcriptional control of gene expression. the lethality of antisense knock-down expression suggests the essential role of this gene family in potatoes.  allelic polymorphism of gsl genes in potato pcr isolation, cloning and sequencing of the coding region from the autotetraploid potato cultivar ‘iwa’ revealed two alleles for the gsl gene and two alleles for the gsl gene. from the frequency of clones with the gsl alleles, it is estimated that iwa has three copies of the a allele and one copy of the a allele. similarly for the gsl gene, iwa has three copies of the b allele and one copy of the b allele. the a and a alleles of the gsl gene differed by single nucleotide polymorphisms and four indels of 1– nucleotides. all of these variant nucleotides were in the introns, except for only three synonymous snps in the exons. similarly, the b and b alleles of the gsl gene were polymorphic for snps and five indels of 1– nucleotides, with only four synonymous snps all occurring in the third exon of gsl pcr isolation and direct sequencing of cdna products determined the sequence of cdna from mature transcripts for both the gsl and gsl genes. alignment of the genomic and cdna sequences confirmed the exon and intron regions in both the gsl and gsl genes. the gsl gene consists of two exons of and nucleotides interrupted by a single intron of nucleotides. the gsl gene is composed of three exons of and nucleotides respectively, interspersed with two introns of and nucleotides. the exon/intron boundaries were identical in all alleles of both the gsl and gsl genes, with the splice sites possessing the conserved 5’gt and 3’ag dinucleotides, consistent with the consensus sequences of the intron at both donor and acceptor sites. structure of full length gsl genes the sequence of the coding regions of the gsl and gsl genes described above were used for interrogation of the potato genome sequence. firstly, gsl and gsl nucleotide sequences were used to blast search the reference cds and genomic sequences to identify gene locations on superscaffold assemblies. secondly, annotated protein sequences from potato genome assembly version of the genotype dm1- r were also searched for the presence of the gasa pfam motif pf. results showed single copy genes for each of gsl and gsl gsl is located on superscaffold pgsc0003dmb which has been mapped to chromosome whereas gsl is located on superscaffold pgsc0003dmb which has been mapped to chromosome. several other genes similar to gsl and gasa genes were also identified; results of these searches are shown in additional file 3: table s the general structure of the gsl and gsl genes was analysed based on the dm sequence of the potato genome. for the gsl gene, up to nucleotides were recovered upstream from the putative transcription start site. we were only able to confirm up to nucleotides upstream of the putative transcription start site for the gsl gene due to a gap in the assembly of the reference potato genome in the upstream region of the gsl promoter. however, we documented at least nucleotides downstream of the stop codon for both gsl genes. a putative transcription start site was predicted based on a plant dimer motif yr rule at nucleotides and nucleotides upstream from the first base of the translation start site for the gsl and gsl genes, respectively. putative cis-elements were identified for the gsl gene. these are tata-box, a pyrimidine patch and caat-box. in gsl a putative tata-box and y patch were located from nucleotides − to − and nucleotides − to − respectively. since we were unable to locate a satisfactory caat-box, we identified a ‘hypothetical’ caat-box in the gsl promoter. next generation sequencing and snp discovery genomic regions for gsl and gsl were further analyzed for sequence variation by aligning re-sequence data generated from illumina reads of the diploid rh89-039- and data generated from four tetraploid lines using the illumina gaiix platform. illumina short insert read pair data were generated for each line. reads were aligned to the reference genome using bwa. alignments were further analysed using samtools, polysnp and visualized using geneious. for the diploid rh and the four tetraploid potato genotypes the structure of the gsl and gsl genes and sequence polymorphisms for the various alleles were annotated manually and compared to the reference dm potato genome. the locations of snps and indels identified across all alleles from all genotypes is illustrated in figure  and summarized in table  in gsl multiple snps and indels were identified within the non-coding regions, with the greatest frequency occurring within the intron and the region from − to − nucleotide positions relative to the 5’utr. the 5’utr contains only a single snp that is found in one re-sequenced genotype. the exon regions have no indels and only very rare synonymous snps, with an overall snp frequency in exons of one snp/ nucleotides. in contrast, the single intron contains snps and different indels. the gsl gene also exhibits the highest snp frequency within the introns. a deletion of nucleotides was found within the 5’utr of dm, but not in the other genotypes. other indels were found in all gene components except the exons. consistent with the gsl gene, the exons of gsl exhibit only very rare synonymous snps among the alleles from all genotypes, with an overall snp frequency in exons of one snp/ nucleotides. gsl and gsl genes the next generation sequence data from four tetraploid potato genotypes, plus the diploid rh, were aligned with the reference potato genome of dm. snp output is from polysnp tool with stringent calling. the rare frequency of snps observed in gsl and gsl within and between the diploid homozygous dm, the diploid heterozygous rh, and the four tetraploid potato genotypes was comparable to other gsl and gasa-like genes, as well as other highly conserved housekeeping genes. the rare snp incidence in these genes across these haplotypes is substantially lower than the one snp every nucleotides observed in a  mb region analyzed for only two rh haplotypes associated with the potato genome sequence, and the one snp every nucleotides and one snp every nucleotides reported by uitdewilligen et al. based on targeted resequencing of tetraploid cultivars. this confirms the highly conserved nature of the gsl and gsl genes, and therefore suggests that they play an essential role in biological function. expression profiles of gsl genes fpkm as expression values for each transcript were extracted from previous data sets representing a range of different potato tissues and treatments. fpkm levels from a total of tissue and treatment libraries from the genotype dm were analyzed for gsl and gsl expression. of the samples analyzed, gsl was expressed at a higher level in samples compared with gsl often by over an order of magnitude in fpkm values. for gsl expression, segura et al. showed by northern analysis that transcripts of stgsl exhibited highest accumulation in stems, shoot apices, young floral buds and petals. similarly, fpkm reads showed highest levels in mature flowers, immature fruit, shoots, petals and carpels. northern analysis also detected expression in tubers and carpels, but not in roots, stolons, leaves, sepals or stamens. in contrast to the northern analysis, fpkm analysis showed that gsl was expressed in roots, stolons, leaves and sepals. northern analysis and fpkm data are in agreement for the absence of gsl expression in stamens; with the fpkm data also showing no expression in mature fruit and callus tissue. northern analysis also established gsl expression is not induced by biotic or abiotic stresses, while the fpkm data show an absence of gsl transcripts in response to heat stress or baba treatments and a slight increase in transcripts in response to bth treatment. analysis of arabidopsis thaliana plants transgenic for gus fusions to the potato gsl promoter revealed gus expression in root vascular tissue, cotyledons, young leaves and floral organs. this analysis of transcriptional control by the gsl promoter is more consistent with the fpkm data than the previously published northern analysis. fpkm values for the gsl gene and northern analysis are in agreement with highest levels of expression being in carpels and petals, and generally high expression in all tissues examined. however, northern analysis did not detect gsl expression in stolons or roots, which is in contrast to the high levels of expression seen in fpkm data. in addition, fpkm analysis indicates that gsl expression is not induced by biotic or abiotic stresses, plant growth regulator treatments or wounding, although slight reductions are observed in gsl transcripts in response to stress induced by heat, salt, mannitol and baba treatments. this is in contrast with northern analysis, where gsl expression responded to biotic stress, was up-regulated by wounding and aba treatments, down regulated in response to ga and showed no response to salinity or drought treatments. the differences between previously published northern analysis for gsl and gsl and the fpkm values in this study may reflect differences in cultivar/ploidy level and growth/treatment conditions. overall, the fpkm data support the conclusion that gsl is a component of the constitutive defense barriers, especially of the storage and reproductive organs. the fpkm analysis supports the same conclusion for gsl which is in contrast to the previous view that gsl is a component of both constitutive and inducible defense barriers. analysis of gsl promoters since the gsl and gsl genes differed markedly in their magnitude and specificity of transcript accumulation, the promoter regions were analysed for motifs using genomatix-matinspector based on place. a total of and different motifs, previously characterised in other studies, were identified in the gsl and gsl promoter regions, respectively. based on the known transcriptional expression of the gsl genes, the putative roles of gsl proteins, the repeated occurrence of motifs, their presence in all potato genotypes, and their relative position in the promoter region, key motifs with potential functional significance were identified for gsl and gsl. gsl promoter region analysis used genomatix-matinspector based on place. the approximate positions of the motif numbers are indicated on figure 1a. aall four tetraploid genotypes contained all motifs. however, allelic polymorphisms involving snps were occasionally observed in some motifs of these genotypes as indicated by c, k, s, and v. #indicates an indel in the motif of at least one allele of the indicated genotype. gsl promoter region analysis used genomatix-matinspector based on place. the approximate positions of the motif numbers are indicated on figure 1b. aall four tetraploid genotypes contained all motifs. however, allelic polymorphisms involving snps were occasionally observed in some motifs of these genotypes as indicated by c, k, s, and v. v# indicates an insertion into the motif in at least one allele of vtn62-33- eight different key motifs were identified in the gsl promoter, which are repeated up to eleven times resulting in a total of motifs. these involve motifs associated with roles for response to disease and biotic stress, abiotic stresses, light induction, and plant development and were found in the genome sequence of dm as well as all four tetraploid genotypes. however, allelic polymorphisms involving disruptions of these motifs were occasionally observed in the tetraploid genotypes. polymorphic snps were observed in seven of the motifs, with polymorphic indels also observed for nine motifs. in the gsl promoter one key motif was identified that occurs five times and ten other key motifs were identified that occur only once. similar to the promoter of the gsl gene, these have known roles associated with biotic stress, abiotic stress, and development. additional motifs present in the gsl promoter are associated with sugar signaling and hormone responses. these fifteen motifs were all observed in the four tetraploid genotypes, although two were observed to be polymorphic for snps and one was polymorphic for an insertion into at least one allele of the genotype vtn62-33-. the conservation of these motifs across the genome of dm and all four re-sequenced tetraploid genotypes substantiates their importance. their presence in the gsl and gsl promoter regions aligns with the transcriptional expression of the respective genes observed by previous northern analysis and/or the fpkm data. the presence of allelic polymorphisms involving sequence disruptions in some of these motifs could be representative of alleles with potentially altered transcriptional expression of the gsl and gsl genes. antisense knockdown of gsl expression using our standard agrobacterium-mediated transformation protocol for potato, we failed to recover any transformants of potato cultivar iwa with antisense constructs for either the gsl or gsl genes. over leaf explants were subjected to agrobacterium-mediated transformation in each of three experiments for both gsl genes using our well established protocol. we would normally expect to recover at least one regenerated transformant per leaf explant for the potato cultivar iwa when selecting for the kanamycin resistance marker gene used on the binary vector. this expected frequency was achieved in concurrent related experiments using the gsl sense constructs. however, a total of only and putative transformed cell colonies were recovered from all three co-cultivation experiments with agrobacterium containing the gsl and gsl antisense constructs, respectively. all potato cell colonies transformed with the antisense constructs failed to continue growth and eventually senesced and died before complete shoots were regenerated. the senescing cultures were sub-cultured onto medium without timentin™ and dna was extracted from those exhibiting no agrobacterium growth. pcr using primers that bridged the lhca promoter and the antisense gsl coding regions confirmed that these cell colonies were transformed with the intended construct prior to their death. the same dna samples failed to amplify pcr products using primers specific to the agrobacterium virg gene. this confirms the absence of agrobacterium cells in the plant tissue that would otherwise compromise the pcr testing of the transformed potato cell colonies. the lethality of antisense knock-down expression of gsl and gsl suggests an essential role of the gsl gene family for potato development. a previous study achieved partial silencing of gsl in potato by expressing an antisense rna under the control of the 35s promoter. this resulted in plants with reduced height and smaller leaves resulting from reduced cell division, changed leaf metabolism and cell wall composition. the lhca st. promoter used in the present study is known to confer higher and more stable transgene expression than the 35s promoter. consequently, the lethality of gsl and gsl antisense knock-down under the control of the lhca promoter is not unexpected given the dramatic phenotypes observed with the partial silencing from the use of the 35s promoter. it is plausible that these gsl or gsl knock-down impacts, resulting from antisense expression driven by either the 35s or the lhca promoters, could also arise by interference in expression of other closely related gsl and gasa genes. the three most closely related genes to gsl show 68-78% identity in exon regions, whereas the identity with all the other related genes was only 42-56%. for gsl the related gsl and gasa genes have only 44-60% identity in exon regions. although this level of identity may be sufficient to trigger a partial knock-down of these related genes, it is unlikely to result in complete knock-down necessary for lethality. therefore, the lethality of gsl and gsl antisense expression under the control of the lhca promoter can be attributed to knock-down of the gsl and gsl genes.  gsl and gsl gibberellin stimulated-like proteins, are cysteine-rich peptides from potato with antimicrobial properties. given their in vitro antimicrobial activity, the gsl and gsl genes are often considered to play important roles in the innate defence against invading microorganisms and/or to be a key determinant during the interaction between plants and pathogens. in other species similar gsl/gasa proteins are hypothesised to play diverse biological roles in several aspects of plant development, plant responses to biotic or abiotic stress through their participation in hormone crosstalk, and redox homeostasis. to further the understanding of the biological roles of gsl proteins, we undertook a thorough analysis of the structure and expression of these genes in potato. we isolated and sequenced the coding regions and cdnas for both gsl and gsl genes from the potato cultivar iwa. this revealed two alleles for the gsl gene and two alleles for the gsl gene. alignment of the genomic and cdna sequences confirmed the exon and intron regions in both the gsl and gsl genes. the gsl gene consists of two exons of and nucleotides interrupted by a single intron of nucleotides. the gsl gene is composed of three exons of and nucleotides respectively, alternating with two introns of and nucleotides. we have also characterised the full length genes for both gsl and gsl using the genome sequence of diploid potato, coupled with further next generation sequencing of four highly heterozygous tetraploid potato genotypes; cultivars summer delight and karaka, and breeding lines 1021/ and vtn62-33- the frequency of snps in gsl and gsl was very low with only one snp every and nucleotides in exon regions of gsl and gsl respectively, similar to other highly conserved housekeeping genes in potato. specific promoter motifs were also highly conserved among multiple alleles representing the haplotypes from dm and the four re-sequenced tetraploid genotypes, suggesting their importance for biological function. analysis of comprehensive rna-seq data substantiated the role of specific promoter motifs in transcriptional control of gene expression. fpkm analysis established that gsl was expressed at a higher level than gsl in out of libraries, often by an order of magnitude. furthermore, both the gsl and gsl genes exhibited constitutive expression that was not up-regulated in response to biotic or abiotic stresses, hormone treatments or wounding. the fpkm analysis did not always agree with previous northern analysis, although closely matched conclusions from the analysis of arabidopsis thaliana plants transgenic for gus fusions to the potato gsl promoter. the gsl and gsl genes from potato are very highly conserved suggesting they contribute to an important biological function. the known antimicrobial activity of the gsl proteins, coupled with the fpkm analysis from rna-seq data, suggests that both genes contribute to the constitutive defence barriers in potatoes. the lethality of antisense knock-down expression of gsl and gsl coupled with the rare incidence of snps in these genes, suggests an essential role for this gene family. these features are consistent with the gsl protein family playing a role in several aspects of plant development and plant defence responses. 
furthermore, both the gsl and gsl genes exhibited constitutive expression that was not up regulated in response to biotic or abiotic stresses, hormone treatments or wounding. analysis of comprehensive rna-seq data substantiated the role of specific promoter motifs in transcriptional control of gene expression. we have characterised the full length genes for both gsl and gsl from diploid and tetraploid potato using the reference genome sequence of potato, coupled with further next generation sequencing of four highly heterozygous tetraploid cultivars. expression analysis based on the frequency of next generation sequence reads established that gsl was expressed at a higher level than gsl in out of tissue and treatment libraries. the known antimicrobial activity of the gsl proteins, coupled with the fpkm analysis from rna-seq data, implies that both genes contribute to the constitutive defence barriers in potatoes. the lethality of antisense knock-down expression of gsl and gsl coupled with the rare incidence of snps in these genes, suggests an essential role for this gene family. similar peptides in other species have been implicated in diverse biological processes and are hypothesised to play a role in several aspects of plant development, plant responses to biotic or abiotic stress through their participation in hormone crosstalk, and redox homeostasis. gsl and gsl gibberellin stimulated-like proteins, are cysteine-rich peptides from potato with antimicrobial properties. potato transformation with antisense knock-down expression cassettes failed to recover viable plants. to help resolve the biological roles of gsl and gsl peptides we have undertaken an in depth analysis of the structure and expression of these genes in potato. the potato gsl and gsl genes are very highly conserved suggesting they contribute to an important biological function. these features are consistent with the gsl protein family playing a role in several aspects of plant development in addition to plant defence against biotic stresses. the frequency of snps in gsl and gsl were very low with only one snp every and nucleotides in exon regions of gsl and gsl respectively.
 gsl and gsl gibberellin stimulated-like proteins, are cysteine-rich peptides from potato with antimicrobial properties. similar peptides in other species have been implicated in diverse biological processes and are hypothesised to play a role in several aspects of plant development, plant responses to biotic or abiotic stress through their participation in hormone crosstalk, and redox homeostasis. to help resolve the biological roles of gsl and gsl peptides we have undertaken an in depth analysis of the structure and expression of these genes in potato.  we have characterised the full length genes for both gsl and gsl from diploid and tetraploid potato using the reference genome sequence of potato, coupled with further next generation sequencing of four highly heterozygous tetraploid cultivars. the frequency of snps in gsl and gsl were very low with only one snp every and nucleotides in exon regions of gsl and gsl respectively. analysis of comprehensive rna-seq data substantiated the role of specific promoter motifs in transcriptional control of gene expression. expression analysis based on the frequency of next generation sequence reads established that gsl was expressed at a higher level than gsl in out of tissue and treatment libraries. furthermore, both the gsl and gsl genes exhibited constitutive expression that was not up regulated in response to biotic or abiotic stresses, hormone treatments or wounding. potato transformation with antisense knock-down expression cassettes failed to recover viable plants.  the potato gsl and gsl genes are very highly conserved suggesting they contribute to an important biological function. the known antimicrobial activity of the gsl proteins, coupled with the fpkm analysis from rna-seq data, implies that both genes contribute to the constitutive defence barriers in potatoes. the lethality of antisense knock-down expression of gsl and gsl coupled with the rare incidence of snps in these genes, suggests an essential role for this gene family. these features are consistent with the gsl protein family playing a role in several aspects of plant development in addition to plant defence against biotic stresses. gsl1gsl2gibberellin stimulated-like proteinspotatosnakingenome analysistranscriptional expression 