BACKGROUND
zebra chip  is an important potato disease causing millions of dollars in losses to both potato producers and processors in the united states  <cit> .
the disease was first discovered in potato fields near saltillo, mexico in  <dig>  and was reported in texas, usa in  <dig>  <cit> .
since  <dig>  the disease has spread to several other states and has been accompanied by serious economic impacts  <cit> .
the disease symptoms are characterized by necrotic flecking and medullary ray discolorations in tubers, leaf chlorosis, twisted stems, swollen nodes, vascular discoloration, leaf scorching, and wilting  <cit> , <cit> .
the putative causal agent of zebra chip disease is ‘candidatus liberibacter solanacearum’  , which is transmitted by the potato psyllid, bactericera cockerelli <cit> , <cit> .
lso has a significantly reduced genome and shares a high degree of similarity with another important plant pathogen, ‘ca.
liberibacter asiaticus’   <cit> , <cit> .
lso is also associated with diseases of other crops including tomato, carrot  in finland, and celery  in spain  <cit> .
currently all commercial potato cultivars appear to be susceptible to lso infection and the only strategy for controlling the spread of the disease is by managing the potato psyllid  <cit> .
plant immunity relies on two levels of defense response against pathogens: pathogen-associated molecular patterns -triggered immunity  that is a component of basal defense and effector-triggered immunity  that reflects immunity to specific strains  <cit> , <cit> .
pti is associated with pamp recognition and activation of plant plasma membrane receptors  <cit> , <cit> .
typically, pti induces several important signaling pathways including calcium ion  influx, reactive oxygen species  production, and mitogen-activated protein kinase  pathway activation  <cit> .
interactions between the peptide flg <dig>  which is located at the n-terminal domain of a flagellin, and host surface-localized pattern recognition receptors flagellin sensing  <dig>  in arabidopsis thaliana, are well studied.
in a. thaliana, fls <dig> interacts with the receptor kinase bak <dig> by forming a functional fls2/bak <dig> complex, which is subsequently followed by a typical pti response  <cit> , <cit> .
orthologs of the arabidopsis fls <dig> have been identified in many plant species including n. benthamiana <cit> , lycopersicum esculentum <cit>  and oryza japonica <cit> .
recently, it has been shown that silencing the expression of nbfls <dig> compromised the expression of downstream genes induced by flg <dig>  <cit> .
in arabidopsis, the ros burst induced by the flg <dig> is regulated by rbohd <cit> .
in n. benthamiana, rbohb, a homolog of rbohd, is essential for ros production and silencing of nbrbohb completely abolishes the ros burst  <cit> , <cit> .
in n. benthamiana, two mitogen-activated protein kinases , salicylic acid-induced protein kinase , and wound-induced protein kinase  are activated quickly after elicitation  <cit> .
furthermore, both sipk and wipk are essential for bacterial immunity in n. benthamiana <cit> .
it has recently been shown that another member of the liberibacter genus, ca.
liberibacter asiaticus , encodes a functional flagellin that partially restored the motility of sinorhizobium meliloti fla mutant and shares characteristics of pathogen-associated molecular patterns  <cit> .
like las, the lso genome contains the same number of open reading frames  that are essential for the structure and assembly of a flagellum  <cit> .
in this study, due to the inability to grow lso in culture, we investigated the pamp activity of lso flagellin  and its peptide flg22lsoin planta via infiltration or agrobacterium-mediated transient expression.
our results demonstrate that transient expression of flalso induces a delayed increase in ros production and slow necrotic cell death in tobacco plants.
although the flg <dig> from p. aeruginosa  induced the typical ros bursts, the flg22lso peptide did not induce ros production in tobacco, tomato, or potato, but did induce callose deposition in these three species.
we further demonstrated that the peptide flg22lso and the flagellin, flalso induced expression of genes associated with pti in n. benthamiana.
these results provide new insights into the role of bacterial flagellin in the development of potato zebra chip disease.
RESULTS
lso encoding a flagellin with a conserved flg22lso peptide
in the lso-zc <dig> genome, several clusters of flagellar biosynthesis related genes were identified by sequence analysis.
ckc_ <dig> was characterized as encoding a flagellin domain-containing protein.
this gene contains  <dig> nucleotides and encodes a  <dig> amino-acid protein, designated as flalso.
flalso shares 61% identity to the flagellin from ca.
liberibacter asiaticus , 59% identity to the flagellin from ca.
liberibacter americanus  and 51% identity to the flagellin from l. crescens bt <dig>  a conserved flagellin domain was identified consisting of  <dig> amino acids located at position  <dig> to  <dig> at the n terminus of flalso and was designated as flg22lso.
flg22lso shares high identity  to the flg <dig> peptides from las, lam and l. crescens bt1and it shares 77% identity to the flg <dig> from the closely related species agrobacterium tumefaciens and sinorhizobium meliloti  <dig>  which do not induce plant immune responses  <cit> , <cit> .
flg22lso shares 41% identity with the flg <dig> from pseudomonas aeruginosa and 55% identity with the flg <dig> from pseudomonas syringae pv.
tabaci, which trigger a strong nonhost innate immune response  <cit> .
in flg <dig>  the amino acid residue d <dig> is critical for its pamp activity  <cit> , <cit> .
although flg22lso possesses amino acid d <dig>  only two other amino acids are conserved in the central rinsakdda motif .
slow necrotic cell death induced by transient expression of the flalso in n. benthamiana
for the transient expression assay of flalso in n. benthamiana, various concentrations of a. tumefaciens gv <dig> with and without flalso were tested.
at an od <dig> of  <dig>  and  <dig> , necrotic cell death was observed in the infiltration zone with pbin:flalso at 8 days after inoculation; the control with pbin vector alone exhibited chlorosis but not necrosis, and gv <dig> with no vector showed no visible response .
in addition, cell death was measured by electrolyte leakage from leaf discs infiltrated with mgcl <dig>  gv <dig> carrying pbin vector and pbin:flalso constructs.
overexpression of the flalso in tobacco leaves increased electrolyte leakage on  <dig> and  <dig> dpi .
taken together, this indicates that the expression of the flagellin gene, flalso, causes necrotic cell death in the infiltrated zone of n. benthamiana.
however, in tomato and potato inoculations, no obvious differences were observed after inoculation with various concentration of gv <dig> containing the pbin vector alone or pbin:flalso, respectively .
callose deposition induced by flg22lso and flg22las
the conserved flagellin domain flg22las from ca.
liberibacter asiaticus induced callose deposition when infiltrated into tobacco  <cit> .
compared to flg22las, flg22lso has three amino acid substitutions including a serine  to alanine  at position  <dig>  and alanine  to serine  substitutions at positions  <dig> and  <dig> .
both synthetic peptides flg22lso and flg22las at a concentration of 40 μm were infiltrated into leaves of n. benthamiana, tomato and potato.
neither peptide induced cell death in the infiltrated zone but callose deposition was observed in infiltrated n. benthamiana, tomato and potato, with more callose deposition from flg22lso compared to flg22las in both tobacco and tomato .
compared to tobacco and potato, a more robust callose deposition was observed in tomato after treatment with either one of the peptides .
ros production induced by the flalso, but not by the flg22lso in n. benthamiana
one of the important events of pti response is a rapid and transient burst of ros production.
we examined whether ros production is induced by flg22lso or flg22las.
neither flg22lso nor flg22las produced an ros burst when infiltrated at  <dig>  μm, 1 μm, 10 μm or 40 μm in tobacco, tomato or potato; while the control flg <dig> from p. aeruginosa shows a typical ros production at a  <dig>  μm concentration in the three plant species .
in contrast, ros production was detected in transient expression of n. benthamiana leaves infiltrated with a. tumefasciens containing pbin:flalso and pbin:flalas clones respectively .
the strongest ros response was observed on the third and fourth day after infiltration.
however, no ros response was detected in tomato or potato infiltrated with these peptides and constructs , which is consistent with the observation that no obvious cell death was induced in these plants.
pti gene expression transiently up-regulated by flg22lso in n. benthamiana
the expression of many plant genes is up-regulated in pamp-triggered immunity  <cit> , <cit> .
to investigate expression levels of pamp-associated genes in n. benthamiana after flg22lso infiltration, reverse transcriptase quantitative pcr  was performed.
after n. benthamiana was infiltrated with the flg22lso peptide, the transcript abundance was measured at  <dig> ,  <dig> ,  <dig> , and  <dig>  hours post infiltration .
the expression of nbfls <dig> was up-regulated approximately  <dig> fold at  <dig>  hpi, more than  <dig> fold at  <dig>  hpi, and then decreased at  <dig>  hpi with flg22lso treatment .
previously, three marker genes in n. benthamiana, nbcyp71d <dig>  nbacre <dig> and nbacre <dig>  were found to rapidly increase following flg22psy treatment  <cit> , <cit> .
these three pamp-marker genes were transiently induced by flg22lso at  <dig>  hpi and then subsequently declined in our rt-qpcr assay, which indicates that flg22lso induces a typical transient pamp triggered immunity gene response.
fls <dig> directly binds bacterial flagellin and then interacts with bak <dig> to form a recognition complex  <cit> .
however, the expression level of nbbak <dig> showed no obvious differences in our assay.
somatic embryogenesis receptor kinase  <dig> /bak <dig> is required for pamp-triggered immunity in n. benthamiana <cit> .
the expression pattern of nbserk3/ nbbak <dig> did not show an obvious change upon flg22lso treatment.
two mapks, nbwipk and nbsipk, were transiently increased at  <dig>  hpi in our experiment.
nbrbohb expression was up-regulated about  <dig> fold except at  <dig>  hpi.
notably, most of these pti related genes are up-regulated at  <dig>  hpi or  <dig>  hpi, and then diminish at  <dig>  hpi.
plastocyanin, which plays a key role in photosynthesis, was reported to be induced in the pti response to nonpathogenic p. fluorescens <cit> .
in our experiment, transcript abundance of nbplastocyanin increased  <dig> fold at  <dig>  hpi and almost  <dig> fold at  <dig>  hpi .
taken together, our results showed that flg22lso transiently induced pamp-triggered gene expression in n. benthamiana.
long lasting pti gene expression induced by flalso in n. benthamiana
the expression of flalso and its elicitation of pamp-associated gene expression in n. benthamiana were investigated after infiltration with a. tumefasciens containing pbin:flalso or the empty pbin vector plasmid.
a high level of expression of flalso was indicated by a strong band from  <dig> to 4 days after inoculation .
a very faint band was observed on  <dig> dpi .
rt-qpcr data further showed that the transcript abundance after inoculation increased to its highest level on  <dig> dpi and decreased to an undetectable level compared to the host reference gene nbef1α .
meanwhile, the gene expression levels of pamp-associated genes were measured at  <dig>   <dig>   <dig>  and  <dig> dpi .
rt-qpcr data showed that the expression of nbfls <dig> was up-regulated more than  <dig> fold at  <dig> dpi.
the abundance of nbbak1/nbserk <dig> was increased about  <dig> fold on  <dig> dpi.
the expression of three other pamp-associated genes, nbcyp71d <dig>  nbacre <dig> and nbacre <dig> were upregulated and varied only slightly at different time after infiltration.
two mapks, especially nbwipk, were up-regulated more than  <dig> fold at  <dig> dpi.
nbrbohb dramatically increased at  <dig> dpi, which agreed with our ros assay results that the strongest ros production was detected at  <dig> dpi.
the expression level of plastocyanin gradually declined over the evaluation period, in contrast to the transient high induction at  <dig>  and  <dig>  hpi observed with flg22lso.
collectively, the expression of pamp-associated genes elicited by the flalso was different from the transient up-regulation with peptide flg22lso induction.
most of these genes were strongly induced at  <dig> dpi, and then decreased, which correlated with the expression pattern of the flalso in n. benthamiana.
discussion
our results revealed that transient expression of the lso flagellin gene  induced a burst of ros and slow necrotic cell death in n. benthamiana plants but not in the tomato or potato plants tested.
although the peptide flg22lso did not induce cell death or a ros reaction, it did induce callose deposition in all three plant species.
we determined that the expression of pamp-triggered genes was transiently up-regulated with synthesized peptide flg22lso treatment.
we further showed that these genes were more strongly affected by agrobacterium-mediated transient expression of the full length gene, flalso.
our results demonstrate that lso flagellin and its short peptide, flg22lso have a pamp activity, and both of them trigger pti response.
callose deposition induced by lso and las peptides
many reports document that flg22lso peptides can induce plant defense responses, including reactive oxygen species , pathogenesis related gene expression and callose deposition  <cit> .
amino acid d <dig> of flg22lso has been demonstrated to be essential for the elicitor activity of xanthomonas and p. syringe pv.
tabaci in non-host species  <cit> , <cit> .
in our study, flg22lso, which contains d <dig>  did not induce visible cell death or ros production in the plants tested, however, it was found to induce callose deposition in tobacco, tomato and potato plants.
this suggests that flg22lso may interact with the fls <dig> receptors of all three plant species.
in addition, we screened over one hundred potato genotypes using flg22lso and flg22las.
we found that flg22lso and flg22las induced ros response in several potato cultivars and the full flagellin gene has the ability to interact with the potato fls <dig> receptor in yeast two hybridization assay .
the flg <dig> peptides from a. tumefaciens and s. meliloti posses the d <dig> amino acid but did not show pamp activity  <cit> .
this indicates that other amino acids are also important for flg <dig> elicitor activity in addition to d <dig>  amino acid changes made at s <dig> and a <dig> completely abolished the pamp activity of flg22las, indicating that serine and aspartate at these positions are essential for flg22las recognition by tobacco plants  <cit> .
compared to flg22las, flg22lso has three amino acid substitutions and induces more callose deposition than flg22las in tobacco and tomato.
it is worth noting that callose deposition was observed although no ros production was detected in the infiltrated plants with either flg22lso or flg22las.
however, callose deposition is sometimes seen in a host plant’s response to pathogen infection as well as being a component of non-host resistance, and high levels of callose are formed in response to las infection in citrus  <cit> .
necrotic cell death induced by the flalso in n. benthamiana
flagellin is a bacterial elicitor associated with the induction of plant and animal defense responses.
in our study, we discovered that transient expression of the flalso induced necrotic cell death in tobacco, which was much slower than that reported for a typical hypersensitive response   <cit> .
transient expression of p. syringae flagellin flic gene with or without signal peptide induced ros and fls2-dependent immunity in a non-host, but did not induce cell death within five days  <cit> .
in our infiltration experiments, the expression of flalso reached the highest level on  <dig> dpi and decreased to a very low level on  <dig> dpi, however necrotic cell death was not observed until 8– <dig> days after infiltration.
this resembles cell death in a compatible pathogen-host interaction, which is much slower than non-host responses.
recently it has been reported that tobacco  is a host for lso  <cit> : response of a weak host rather than a non-host may explain our observation of necrotic cell death rather than a hr, which is characterized as rapid, localized plant cell death  <cit> .
in contrast, perception of pamps leads to small metabolic changes during pti so that energy production can be sustained in the presence of pamps  <cit> .
it is intriguing that transient expression of the full length flalso inside plant cells could also induce up-regulation of pamp-associated gene expression, ros response and slow necrotic cell death.
fls <dig> is a receptor that is known to recognize extracellular flagellin.
in mammals, pamps such as flagellin in the cytosol induce a localized cell-death-associated immune response to pathogens.
however, no cytoplasmic nucleotide-binding leucine-rich  receptor or other cytoplasmic receptor was identified in plants  <cit> .
most studies on plant fls <dig> have focused on interaction with flg <dig> of flagellin administered extracellularly  <cit> , as would occur with infection by pseudomonas and many other bacterial pathogens.
candidatus liberibacter species are strictly intracellular, and therefore, in order to function as a pamp, their flagellins would require recognition within the plant cell.
it has been demonstrated that transgenic expression of the flagellin from a rice-incompatible strain of acidovorax avenae induced immune responses, expression of defense related genes, production of hydrogen peroxide and cell death in rice plants  <cit> .
in another study, when fls2-gfp was transgenically expressed, the fls <dig> was primarily localized on the cell membrane before binding to flg <dig>  after binding, the fls <dig> accumulated into intracellular mobile vesicles and degraded upon flg <dig> activation  <cit> .
in addition to being localized on cell membranes, fls <dig> was also observed in intracellular vesicles of various sizes and shapes in a protoplast assay  <cit> .
all of these together indicate that there is a unique pathway involving the interaction between fls <dig> and lso flagellin inside a plant cell.
further research is necessary to understand this pathway and downstream host response.
pti gene expression responses induced by lso flagellin and peptide
as a complement to the observations discussed above, we investigated flg22lso-induced gene expression following infiltration into n. benthamiana.
nbfls <dig> and three other pamp-marker genes, nbcyp71d <dig>  nbacre <dig> and nbacre <dig>  were shown to be rapidly and transiently up-regulated  as was previously reported with the flg <dig> from pseudomonas <cit> , <cit> .
we also assessed several other genes which are reported to be associated with different host-defense systems.
acre  genes associated with race-specific defense responses were found to be up-regulated by the infiltration of flg22lso in n. benthamiana, similar to the result seen when resistant-race tobacco cells  are treated with the fungal elicitor avr <dig>  <cit> .
when p. fluorescens was infiltrated into non-host n. benthamiana, the plastocyanin gene was markedly up-regulated, and silencing this gene compromised pti  <cit> .
plastocyanin is a small cu-containing protein which acts as an electron carrier between the cytochrome b6f and photosystem  <dig> complexes in the photosynthetic electron-transfer chain  <cit> .
surprisingly, our results showed that the expression of nbplastocyanin was the most highly induced gene in the pti response by flg22lso treatment.
however, the up-regulation of nbplastocyanin expression gradually decreased over several days with agrobacterium-mediated expression of full length flalso, which may have resulted from impaired chloroplast function as cell death was slowly induced.
it has been reported that there is cross-talk between pamp-triggered immunity and photosynthesis  <cit> .
though a typical pattern of pamp-triggered gene expression was observed with flg22lso infiltration, no ros production was detected.
this may be explained by the fact that two different signaling pathways were separated after calcium influx initiation: one leading to the ros burst and the other to mapks and other gene activation  <cit> .
it was reported that domains other than flg <dig> contribute very little to the elicitation of the fls2-mediated arabidopsis defense response  <cit> .
however, a second region designated as flgii- <dig> within flagellin apart from flg <dig> was recently identified in p. syringae pv.
tomato and flgii- <dig> was shown to induce ros in n. benthamiana not in arabidopsis or bean  <cit> .
furthermore, the allelic variation of flg <dig> and flgii- <dig> were reported to affect the plant immune response significantly, and to have no effect on bacterial motility  <cit> .
this may explain our findings that the full flagellin gene, flalso, not the peptide flg22lso, has the ability to induce ros production and slow cell death in tobacco, which suggests other flagellin domains such as flgii- <dig> or post-translational modifications may also be associated with plant defense response.
it is important to point out that transient expression of the flalso only induced ros production in tobacco but did not lead to ros reaction in tested tomato and potato plants.
this could be explained by the sequence variations within the fls <dig> genes among species/varieties of host plants .
it is established that fls <dig> directly binds to flagellin in the pseudomonas non-host system  <cit>  and then interacts with bak <dig> to form the fls2/bak <dig> complex, which activates the downstream signaling such as mapk pathway.
in our experiments, the expression of two mapks, nbwipk and nbsipk were indeed up-regulated in n. benthamiana by the flalso .
the prolonged activation of a mapk pathway in cells may cause a redox imbalance and generate ros, eventually leading to cell death  <cit> .
since map kinases are primarily regulated at protein levels, the levels of these proteins in n. benthamiana after inoculation with flg22lso and or flalso need a further investigation.
pamp-triggered immunity is important for plants to limit pathogen growth or generate signals for adaptation to secondary infections  <cit> .
pamps, including flg <dig>  activate components of the salicylic acid and jasmonic acid defense pathways, which protect against potential pathogenic bacteria  <cit> .
the molecular events that occur during pti and elicitor-triggered immunity  partially overlap including sa, ros, and activation of mapk cascades  <cit> .
however, eti elicits a much stronger response than pti, indicating a quantitative difference between these two immunity responses  <cit> .
the discovery that the flalso and flg22lso have pamp activity and trigger pti reveals that the flagellin from intracellular bacteria can initiate plant defense responses.
the identification of a compatible fls <dig> is critical for the development of potato plants with increased resistance against ca.
l. solanacearum via marker-assisted conventional breeding and genetic engineering.
CONCLUSIONS
zebra chip  is an important potato disease associated with the phloem-limited intracellular bacterium ‘candidatus liberibacter solanacearum’ .
in this study, we examined the pamp activity of the flagellin of lso and its functional domain, flg22lsoin planta.
we found that flg22lso has the ability to induce callose deposition and it also triggers transient up-regulation of pti associated genes in n. benthamiana.
we determined that the expression of nbfls <dig> and three marker genes, nbcyp71d <dig>  nbacre <dig> and nbacre <dig>  are rapidly upregulated.
surprisingly we found the expression of nbplastocyanin increased dramatically, rising by  <dig> fold at  <dig> hpi.
however, neither cell death nor ros were induced by the flg22lso.
we also determined that expression of the full length flagellin gene induces a much stronger pti response compared to peptide flg22lso, especially upregulation of the pamp-associated genes nbfls <dig> and nbrbohb.
in addition, the expression of flalso induced ros production and necrotic cell death in n. benthamiana via agrobacterium-mediated transient expression.
the discovery that the flalso and its short peptide have pamp activity and the ability to trigger pti provide new insights into the role of bacterial flagellin in the development of potato zebra chip disease and a potential application in breeding for resistance.

 zebra chip is an important potato disease causing millions of dollars in losses to both potato producers and processors in the united states. the disease was first discovered in potato fields near saltillo, mexico in and was reported in texas, usa in. since the disease has spread to several other states and has been accompanied by serious economic impacts. the disease symptoms are characterized by necrotic flecking and medullary ray discolorations in tubers, leaf chlorosis, twisted stems, swollen nodes, vascular discoloration, leaf scorching, and wilting,. the putative causal agent of zebra chip disease is ‘candidatus liberibacter solanacearum’, which is transmitted by the potato psyllid, bactericera cockerelli,. lso has a significantly reduced genome and shares a high degree of similarity with another important plant pathogen, ‘ca. liberibacter asiaticus’,. lso is also associated with diseases of other crops including tomato, carrot in finland, and celery in spain. currently all commercial potato cultivars appear to be susceptible to lso infection and the only strategy for controlling the spread of the disease is by managing the potato psyllid. plant immunity relies on two levels of defense response against pathogens: pathogen-associated molecular patterns -triggered immunity that is a component of basal defense and effector-triggered immunity that reflects immunity to specific strains,. pti is associated with pamp recognition and activation of plant plasma membrane receptors,. typically, pti induces several important signaling pathways including calcium ion influx, reactive oxygen species production, and mitogen-activated protein kinase pathway activation. interactions between the peptide flg which is located at the n-terminal domain of a flagellin, and host surface-localized pattern recognition receptors flagellin sensing in arabidopsis thaliana, are well studied. in a. thaliana, fls interacts with the receptor kinase bak by forming a functional fls2/bak complex, which is subsequently followed by a typical pti response,. orthologs of the arabidopsis fls have been identified in many plant species including n. benthamiana, lycopersicum esculentum and oryza japonica. recently, it has been shown that silencing the expression of nbfls compromised the expression of downstream genes induced by flg. in arabidopsis, the ros burst induced by the flg is regulated by rbohd. in n. benthamiana, rbohb, a homolog of rbohd, is essential for ros production and silencing of nbrbohb completely abolishes the ros burst,. in n. benthamiana, two mitogen-activated protein kinases, salicylic acid-induced protein kinase, and wound-induced protein kinase are activated quickly after elicitation. furthermore, both sipk and wipk are essential for bacterial immunity in n. benthamiana. it has recently been shown that another member of the liberibacter genus, ca. liberibacter asiaticus, encodes a functional flagellin that partially restored the motility of sinorhizobium meliloti fla mutant and shares characteristics of pathogen-associated molecular patterns. like las, the lso genome contains the same number of open reading frames that are essential for the structure and assembly of a flagellum. in this study, due to the inability to grow lso in culture, we investigated the pamp activity of lso flagellin and its peptide flg22lsoin planta via infiltration or agrobacterium-mediated transient expression. our results demonstrate that transient expression of flalso induces a delayed increase in ros production and slow necrotic cell death in tobacco plants. although the flg from p. aeruginosa induced the typical ros bursts, the flg22lso peptide did not induce ros production in tobacco, tomato, or potato, but did induce callose deposition in these three species. we further demonstrated that the peptide flg22lso and the flagellin, flalso induced expression of genes associated with pti in n. benthamiana. these results provide new insights into the role of bacterial flagellin in the development of potato zebra chip disease.  lso encoding a flagellin with a conserved flg22lso peptide in the lso-zc genome, several clusters of flagellar biosynthesis related genes were identified by sequence analysis. ckc_ was characterized as encoding a flagellin domain-containing protein. this gene contains nucleotides and encodes a amino-acid protein, designated as flalso. flalso shares 61% identity to the flagellin from ca. liberibacter asiaticus, 59% identity to the flagellin from ca. liberibacter americanus and 51% identity to the flagellin from l. crescens bt a conserved flagellin domain was identified consisting of amino acids located at position to at the n terminus of flalso and was designated as flg22lso. flg22lso shares high identity to the flg peptides from las, lam and l. crescens bt1and it shares 77% identity to the flg from the closely related species agrobacterium tumefaciens and sinorhizobium meliloti which do not induce plant immune responses,. flg22lso shares 41% identity with the flg from pseudomonas aeruginosa and 55% identity with the flg from pseudomonas syringae pv. tabaci, which trigger a strong nonhost innate immune response. in flg the amino acid residue d is critical for its pamp activity,. although flg22lso possesses amino acid d only two other amino acids are conserved in the central rinsakdda motif. slow necrotic cell death induced by transient expression of the flalso in n. benthamiana for the transient expression assay of flalso in n. benthamiana, various concentrations of a. tumefaciens gv with and without flalso were tested. at an od of and, necrotic cell death was observed in the infiltration zone with pbin:flalso at 8 days after inoculation; the control with pbin vector alone exhibited chlorosis but not necrosis, and gv with no vector showed no visible response. in addition, cell death was measured by electrolyte leakage from leaf discs infiltrated with mgcl gv carrying pbin vector and pbin:flalso constructs. overexpression of the flalso in tobacco leaves increased electrolyte leakage on and dpi. taken together, this indicates that the expression of the flagellin gene, flalso, causes necrotic cell death in the infiltrated zone of n. benthamiana. however, in tomato and potato inoculations, no obvious differences were observed after inoculation with various concentration of gv containing the pbin vector alone or pbin:flalso, respectively. callose deposition induced by flg22lso and flg22las the conserved flagellin domain flg22las from ca. liberibacter asiaticus induced callose deposition when infiltrated into tobacco. compared to flg22las, flg22lso has three amino acid substitutions including a serine to alanine at position and alanine to serine substitutions at positions and. both synthetic peptides flg22lso and flg22las at a concentration of 40 μm were infiltrated into leaves of n. benthamiana, tomato and potato. neither peptide induced cell death in the infiltrated zone but callose deposition was observed in infiltrated n. benthamiana, tomato and potato, with more callose deposition from flg22lso compared to flg22las in both tobacco and tomato. compared to tobacco and potato, a more robust callose deposition was observed in tomato after treatment with either one of the peptides. ros production induced by the flalso, but not by the flg22lso in n. benthamiana one of the important events of pti response is a rapid and transient burst of ros production. we examined whether ros production is induced by flg22lso or flg22las. neither flg22lso nor flg22las produced an ros burst when infiltrated at  μm, 1 μm, 10 μm or 40 μm in tobacco, tomato or potato; while the control flg from p. aeruginosa shows a typical ros production at a  μm concentration in the three plant species. in contrast, ros production was detected in transient expression of n. benthamiana leaves infiltrated with a. tumefasciens containing pbin:flalso and pbin:flalas clones respectively. the strongest ros response was observed on the third and fourth day after infiltration. however, no ros response was detected in tomato or potato infiltrated with these peptides and constructs, which is consistent with the observation that no obvious cell death was induced in these plants. pti gene expression transiently up-regulated by flg22lso in n. benthamiana the expression of many plant genes is up-regulated in pamp-triggered immunity,. to investigate expression levels of pamp-associated genes in n. benthamiana after flg22lso infiltration, reverse transcriptase quantitative pcr was performed. after n. benthamiana was infiltrated with the flg22lso peptide, the transcript abundance was measured at,,, and  hours post infiltration. the expression of nbfls was up-regulated approximately fold at hpi, more than fold at hpi, and then decreased at hpi with flg22lso treatment. previously, three marker genes in n. benthamiana, nbcyp71d nbacre and nbacre were found to rapidly increase following flg22psy treatment,. these three pamp-marker genes were transiently induced by flg22lso at hpi and then subsequently declined in our rt-qpcr assay, which indicates that flg22lso induces a typical transient pamp triggered immunity gene response. fls directly binds bacterial flagellin and then interacts with bak to form a recognition complex. however, the expression level of nbbak showed no obvious differences in our assay. somatic embryogenesis receptor kinase /bak is required for pamp-triggered immunity in n. benthamiana. the expression pattern of nbserk3/ nbbak did not show an obvious change upon flg22lso treatment. two mapks, nbwipk and nbsipk, were transiently increased at hpi in our experiment. nbrbohb expression was up-regulated about fold except at hpi. notably, most of these pti related genes are up-regulated at hpi or hpi, and then diminish at hpi. plastocyanin, which plays a key role in photosynthesis, was reported to be induced in the pti response to nonpathogenic p. fluorescens. in our experiment, transcript abundance of nbplastocyanin increased fold at hpi and almost fold at hpi. taken together, our results showed that flg22lso transiently induced pamp-triggered gene expression in n. benthamiana. long lasting pti gene expression induced by flalso in n. benthamiana the expression of flalso and its elicitation of pamp-associated gene expression in n. benthamiana were investigated after infiltration with a. tumefasciens containing pbin:flalso or the empty pbin vector plasmid. a high level of expression of flalso was indicated by a strong band from to 4 days after inoculation. a very faint band was observed on dpi. rt-qpcr data further showed that the transcript abundance after inoculation increased to its highest level on dpi and decreased to an undetectable level compared to the host reference gene nbef1α. meanwhile, the gene expression levels of pamp-associated genes were measured at and dpi. rt-qpcr data showed that the expression of nbfls was up-regulated more than fold at dpi. the abundance of nbbak1/nbserk was increased about fold on dpi. the expression of three other pamp-associated genes, nbcyp71d nbacre and nbacre were upregulated and varied only slightly at different time after infiltration. two mapks, especially nbwipk, were up-regulated more than fold at dpi. nbrbohb dramatically increased at dpi, which agreed with our ros assay results that the strongest ros production was detected at dpi. the expression level of plastocyanin gradually declined over the evaluation period, in contrast to the transient high induction at and hpi observed with flg22lso. collectively, the expression of pamp-associated genes elicited by the flalso was different from the transient up-regulation with peptide flg22lso induction. most of these genes were strongly induced at dpi, and then decreased, which correlated with the expression pattern of the flalso in n. benthamiana. discussion our results revealed that transient expression of the lso flagellin gene induced a burst of ros and slow necrotic cell death in n. benthamiana plants but not in the tomato or potato plants tested. although the peptide flg22lso did not induce cell death or a ros reaction, it did induce callose deposition in all three plant species. we determined that the expression of pamp-triggered genes was transiently up-regulated with synthesized peptide flg22lso treatment. we further showed that these genes were more strongly affected by agrobacterium-mediated transient expression of the full length gene, flalso. our results demonstrate that lso flagellin and its short peptide, flg22lso have a pamp activity, and both of them trigger pti response. callose deposition induced by lso and las peptides many reports document that flg22lso peptides can induce plant defense responses, including reactive oxygen species, pathogenesis related gene expression and callose deposition. amino acid d of flg22lso has been demonstrated to be essential for the elicitor activity of xanthomonas and p. syringe pv. tabaci in non-host species,. in our study, flg22lso, which contains d did not induce visible cell death or ros production in the plants tested, however, it was found to induce callose deposition in tobacco, tomato and potato plants. this suggests that flg22lso may interact with the fls receptors of all three plant species. in addition, we screened over one hundred potato genotypes using flg22lso and flg22las. we found that flg22lso and flg22las induced ros response in several potato cultivars and the full flagellin gene has the ability to interact with the potato fls receptor in yeast two hybridization assay. the flg peptides from a. tumefaciens and s. meliloti posses the d amino acid but did not show pamp activity. this indicates that other amino acids are also important for flg elicitor activity in addition to d amino acid changes made at s and a completely abolished the pamp activity of flg22las, indicating that serine and aspartate at these positions are essential for flg22las recognition by tobacco plants. compared to flg22las, flg22lso has three amino acid substitutions and induces more callose deposition than flg22las in tobacco and tomato. it is worth noting that callose deposition was observed although no ros production was detected in the infiltrated plants with either flg22lso or flg22las. however, callose deposition is sometimes seen in a host plant’s response to pathogen infection as well as being a component of non-host resistance, and high levels of callose are formed in response to las infection in citrus. necrotic cell death induced by the flalso in n. benthamiana flagellin is a bacterial elicitor associated with the induction of plant and animal defense responses. in our study, we discovered that transient expression of the flalso induced necrotic cell death in tobacco, which was much slower than that reported for a typical hypersensitive response. transient expression of p. syringae flagellin flic gene with or without signal peptide induced ros and fls2-dependent immunity in a non-host, but did not induce cell death within five days. in our infiltration experiments, the expression of flalso reached the highest level on dpi and decreased to a very low level on dpi, however necrotic cell death was not observed until 8– days after infiltration. this resembles cell death in a compatible pathogen-host interaction, which is much slower than non-host responses. recently it has been reported that tobacco is a host for lso: response of a weak host rather than a non-host may explain our observation of necrotic cell death rather than a hr, which is characterized as rapid, localized plant cell death. in contrast, perception of pamps leads to small metabolic changes during pti so that energy production can be sustained in the presence of pamps. it is intriguing that transient expression of the full length flalso inside plant cells could also induce up-regulation of pamp-associated gene expression, ros response and slow necrotic cell death. fls is a receptor that is known to recognize extracellular flagellin. in mammals, pamps such as flagellin in the cytosol induce a localized cell-death-associated immune response to pathogens. however, no cytoplasmic nucleotide-binding leucine-rich receptor or other cytoplasmic receptor was identified in plants. most studies on plant fls have focused on interaction with flg of flagellin administered extracellularly, as would occur with infection by pseudomonas and many other bacterial pathogens. candidatus liberibacter species are strictly intracellular, and therefore, in order to function as a pamp, their flagellins would require recognition within the plant cell. it has been demonstrated that transgenic expression of the flagellin from a rice-incompatible strain of acidovorax avenae induced immune responses, expression of defense related genes, production of hydrogen peroxide and cell death in rice plants. in another study, when fls2-gfp was transgenically expressed, the fls was primarily localized on the cell membrane before binding to flg after binding, the fls accumulated into intracellular mobile vesicles and degraded upon flg activation. in addition to being localized on cell membranes, fls was also observed in intracellular vesicles of various sizes and shapes in a protoplast assay. all of these together indicate that there is a unique pathway involving the interaction between fls and lso flagellin inside a plant cell. further research is necessary to understand this pathway and downstream host response. pti gene expression responses induced by lso flagellin and peptide as a complement to the observations discussed above, we investigated flg22lso-induced gene expression following infiltration into n. benthamiana. nbfls and three other pamp-marker genes, nbcyp71d nbacre and nbacre were shown to be rapidly and transiently up-regulated as was previously reported with the flg from pseudomonas,. we also assessed several other genes which are reported to be associated with different host-defense systems. acre genes associated with race-specific defense responses were found to be up-regulated by the infiltration of flg22lso in n. benthamiana, similar to the result seen when resistant-race tobacco cells are treated with the fungal elicitor avr. when p. fluorescens was infiltrated into non-host n. benthamiana, the plastocyanin gene was markedly up-regulated, and silencing this gene compromised pti. plastocyanin is a small cu-containing protein which acts as an electron carrier between the cytochrome b6f and photosystem complexes in the photosynthetic electron-transfer chain. surprisingly, our results showed that the expression of nbplastocyanin was the most highly induced gene in the pti response by flg22lso treatment. however, the up-regulation of nbplastocyanin expression gradually decreased over several days with agrobacterium-mediated expression of full length flalso, which may have resulted from impaired chloroplast function as cell death was slowly induced. it has been reported that there is cross-talk between pamp-triggered immunity and photosynthesis. though a typical pattern of pamp-triggered gene expression was observed with flg22lso infiltration, no ros production was detected. this may be explained by the fact that two different signaling pathways were separated after calcium influx initiation: one leading to the ros burst and the other to mapks and other gene activation. it was reported that domains other than flg contribute very little to the elicitation of the fls2-mediated arabidopsis defense response. however, a second region designated as flgii- within flagellin apart from flg was recently identified in p. syringae pv. tomato and flgii- was shown to induce ros in n. benthamiana not in arabidopsis or bean. furthermore, the allelic variation of flg and flgii- were reported to affect the plant immune response significantly, and to have no effect on bacterial motility. this may explain our findings that the full flagellin gene, flalso, not the peptide flg22lso, has the ability to induce ros production and slow cell death in tobacco, which suggests other flagellin domains such as flgii- or post-translational modifications may also be associated with plant defense response. it is important to point out that transient expression of the flalso only induced ros production in tobacco but did not lead to ros reaction in tested tomato and potato plants. this could be explained by the sequence variations within the fls genes among species/varieties of host plants. it is established that fls directly binds to flagellin in the pseudomonas non-host system and then interacts with bak to form the fls2/bak complex, which activates the downstream signaling such as mapk pathway. in our experiments, the expression of two mapks, nbwipk and nbsipk were indeed up-regulated in n. benthamiana by the flalso. the prolonged activation of a mapk pathway in cells may cause a redox imbalance and generate ros, eventually leading to cell death. since map kinases are primarily regulated at protein levels, the levels of these proteins in n. benthamiana after inoculation with flg22lso and or flalso need a further investigation. pamp-triggered immunity is important for plants to limit pathogen growth or generate signals for adaptation to secondary infections. pamps, including flg activate components of the salicylic acid and jasmonic acid defense pathways, which protect against potential pathogenic bacteria. the molecular events that occur during pti and elicitor-triggered immunity partially overlap including sa, ros, and activation of mapk cascades. however, eti elicits a much stronger response than pti, indicating a quantitative difference between these two immunity responses. the discovery that the flalso and flg22lso have pamp activity and trigger pti reveals that the flagellin from intracellular bacteria can initiate plant defense responses. the identification of a compatible fls is critical for the development of potato plants with increased resistance against ca. l. solanacearum via marker-assisted conventional breeding and genetic engineering.  zebra chip is an important potato disease associated with the phloem-limited intracellular bacterium ‘candidatus liberibacter solanacearum’. in this study, we examined the pamp activity of the flagellin of lso and its functional domain, flg22lsoin planta. we found that flg22lso has the ability to induce callose deposition and it also triggers transient up-regulation of pti associated genes in n. benthamiana. we determined that the expression of nbfls and three marker genes, nbcyp71d nbacre and nbacre are rapidly upregulated. surprisingly we found the expression of nbplastocyanin increased dramatically, rising by fold at hpi. however, neither cell death nor ros were induced by the flg22lso. we also determined that expression of the full length flagellin gene induces a much stronger pti response compared to peptide flg22lso, especially upregulation of the pamp-associated genes nbfls and nbrbohb. in addition, the expression of flalso induced ros production and necrotic cell death in n. benthamiana via agrobacterium-mediated transient expression. the discovery that the flalso and its short peptide have pamp activity and the ability to trigger pti provide new insights into the role of bacterial flagellin in the development of potato zebra chip disease and a potential application in breeding for resistance. 
our findings provide new insights into the role of the lso flagellin in the development of potato zebra chip disease and potential application in breeding for resistance. our results showed that the expression of flalso via agrobacterium-mediated transient expression induced a slow necrotic cell death in the inoculated leaves of n. benthamiana, which was coupled with a burst of reactive oxygen species production. the flagellin of lso and its functional domain, flg22lso share characteristics of pathogen-associated molecular patterns, and trigger unique innate immune responses in n. benthamiana. moreover, the expression of several representative genes involved in innate immunity was transiently up-regulated by the flg22lso in n. benthamiana. the flalso, however, induced stronger up-regulation of these representative genes compared to the flg22lso, especially that of flagellin receptor flagellin sensing and respiratory burst oxidase in n. benthamiana. although neither cell death nor ros were induced by the synthetic flg22lso, a weak callose deposition was observed in infiltrated leaves of tobacco, tomato, and potato plants. candidatus liberibacter solanacearumflagellinflg22cell deathreactive oxygen speciesgene expression slow and weak activation of the innate immune response in host plants by the flagellin of lso may reflect the nature of its intracellular life cycle. to understand the innate immune responses triggered by this unculturable intracellular bacterium, we studied the pathogen-associated molecular patterns that triggered immunity in nicotiana benthamiana, using the flg22lso peptide and the full length flalso gene. ‘candidatus liberibacter solanacearum’ is a phloem-limited alphaproteobacterium associated with the devastating zebra chip disease of potato.
 ‘candidatus liberibacter solanacearum’ is a phloem-limited alphaproteobacterium associated with the devastating zebra chip disease of potato. like other members of liberibacter, lso-zc encodes a flagellin domain-containing protein with a conserved amino-acid peptide. to understand the innate immune responses triggered by this unculturable intracellular bacterium, we studied the pathogen-associated molecular patterns that triggered immunity in nicotiana benthamiana, using the flg22lso peptide and the full length flalso gene.  our results showed that the expression of flalso via agrobacterium-mediated transient expression induced a slow necrotic cell death in the inoculated leaves of n. benthamiana, which was coupled with a burst of reactive oxygen species production. moreover, the expression of several representative genes involved in innate immunity was transiently up-regulated by the flg22lso in n. benthamiana. the flalso, however, induced stronger up-regulation of these representative genes compared to the flg22lso, especially that of flagellin receptor flagellin sensing and respiratory burst oxidase in n. benthamiana. although neither cell death nor ros were induced by the synthetic flg22lso, a weak callose deposition was observed in infiltrated leaves of tobacco, tomato, and potato plants.  the flagellin of lso and its functional domain, flg22lso share characteristics of pathogen-associated molecular patterns, and trigger unique innate immune responses in n. benthamiana. slow and weak activation of the innate immune response in host plants by the flagellin of lso may reflect the nature of its intracellular life cycle. our findings provide new insights into the role of the lso flagellin in the development of potato zebra chip disease and potential application in breeding for resistance. candidatus liberibacter solanacearumflagellinflg22cell deathreactive oxygen speciesgene expression 