BACKGROUND
mitochondrial alternative oxidases  are important components of the alternative respiratory pathway of plants  <cit> ; aox genes have been isolated from several important plant species  <cit> .
synthesis of aox can be induced when the cytochrome pathway is inhibited, or when the plant is wounded, treated with ethylene, cycloheximide, chloramphenicol, or if the plant is exposed to cold environmental conditions  <cit> .
in addition, the aox pathway can also be induced by treatments with salicylic acid   <cit> , nitric oxide  <cit> , reactive oxygen species  <cit> , high light intensities  <cit>  or pathogen challenge.
because sa induction has been linked to the defense response in plants, it has been suggested that the alternative pathway might be associated with disease resistance in plants  <cit>  including resistance to viruses  <cit> .
evidence supporting this hypothesis includes the finding that elevated levels of aox in tobacco inhibit long-distance movement of cucumber mosaic virus  and replication of tobacco mosaic virus  and potato virus x   <cit> .
furthermore, additional works with cytochrome inhibitors and salicylhydroxamic acid  have led to the proposal that the aox pathway and the products of the aox genes play a key role in the resistance of tobacco plants to virus infection  <cit> .
other studies have suggested, however, that aox is not a critical component of plant viral resistance but that it may play a role in the development of the hypersensitive response  <cit> .
elevated aox gene expression levels had no clear-cut effects on sa-induced resistance to systemic infection by tmv in transgenic tobacco.
moreover, resistance to tmv in tobacco induced by antimycin a , an inhibitor of the cytochrome pathway, was repressed with increased alternative pathway capacity, and both sa- and aa-induced resistances were enhanced when alternative pathway capacity was reduced  <cit> .
furthermore, high-levels of alternative oxidase expression allowed increased tmv spread and the development of severe symptoms in nn-type tobacco and nicotiana benthamiana  <cit> .
the involvement of aox in virus resistance has been reported in a limited number of plant species and virus combinations, however, the mechanisms of this antiviral action varied  <cit> .
in order to accumulate more evidence that might further elucidate the association of aox with antiviral activity, we generated transgenic tomato and petunia lines with altered aox expression levels and evaluated their resistant levels to tomato spotted wilt virus .
RESULTS
tomato and petunia transformation and controlled pollination of transgenic lines
tomato cultivar 'healani' and petunia cultivar 'sheer madness' were transformed with the leaox1au gene isolated from tomato to obtain transgenic lines.
primers specific for the camv 35s promoter and 3'-end of the target inserts pbilaf and pbilac were used in pcr amplifications to detect the presence of integrated leaox1au sequences.
both wild type and transgenic tomato plants contain the same alternative oxidase gene in their genomes, therefore the camv 35s promoter fragment dna , was used to probe ecori-digested genomic dna.
a single ecor i recognition site is located near the left border of the inserted sequences, thus each band shown in figure  <dig> should represent different insert locations.
tomato lines cdt <dig> and ckt <dig> showed single bands, and may represent single copy transformants.
the other  <dig> lines showed  <dig> to  <dig> bands, indicating that these  <dig> lines were transformed with multiple copies of the leaox1au construct.
several putatively transgenic tomato lines were analyzed for aox rna expression by northern analyses using a leaox1-au specific orf dna fragment labeled with dig as a probe.
leaox1-au was not detected in vector only  or non-transgenic  tomato plants .
expression of aox could not be detected in lines cdt <dig>  cdt <dig>  cdt17- <dig> and cdt17- <dig>  the remaining eleven transformed lines showed altered expression of leaox-1au ranging from  <dig>  to  <dig>  times the average signal intensity of the aox orf verses that of 18s rrna.
no clear relationship between the aox copy number determined by southern analyses and the aox rna expression level determined by northern analyses was observed .
the putative transgenic tomato lines were tested for their aox protein expression levels by western blot analyses.
aox expression in non-transgenic and vector-only transformed tomato plants was below detection levels .
thirty-two transgenic tomato lines showed increased expression of aox compared to aox expression levels in non-transgenic and vector-only transgenic tomato plants.
however three transgenic lines  did not show increased aox expression .
based on pcr, southern, northern, and western blot analyses, a total of  <dig> tomato and  <dig> petunia lines were shown to be transformed with the leaox1au gene.
the transgenic lines were self-pollinated and the harvested seeds were dried and stored at 4°c.
seeds of  <dig> tomato lines and  <dig> petunia lines were germinated and grown in the greenhouse.
when the tomato seedlings reached  <dig> to  <dig> cm in height, one leaf disk was collected from each plant using # <dig> cork borer.
transgene constructs in the progeny were confirmed by pcr and southern hybridizations.
only confirmed transgenic lines were analyzed further by tswv challenge.
response of r <dig> generation transgenic tomato plants to tswv infection
twenty to  <dig> r1generation tomato plants from each line were evaluated for resistance to tswv .
within these groups,  <dig> to  <dig> lines were confirmed to contain the transgene by pcr analyses.
the results showed that within each line, all of the transgenic plants had lower elisa readings after tswv challenge than those of non-transgenic plants, indicating that the transgenic tomato plants had elevated resistance level to tswv infection  <cit> .
although no lines were found to be completely immune to tswv, paired t-tests indicated that the progeny from line fkt4- <dig> showed significantly lower elisa values than the wild-type control line.
progeny from seven other transgenic lines did not have statistically significant enhanced resistance compared with wild-type controls, which might possibly be related to positional effects of the insertion event or if the transgene interacted somehow with other genes.
* indicates significant  difference
response of transgenic tomato plants to tswv infection at different time points
to evaluate the dynamic response to tswv infection,  <dig> to  <dig> plants of transgenic lines fkt <dig>  fkt4- <dig>  and wild-type controls were evaluated over time after virus challenge.
tissues were sampled and elisa analyses were performed up to  <dig> days after the second tswv inoculation.
the data in figure  <dig> are the average of three elisa values from 4- <dig> plants of each line.
results show that plants of both transgenic lines and the control all developed tswv infections by  <dig> days after challenge, which showed small dark spots, bronzed leaves that rolled upward, and dieback of young branches.
line fkt <dig> was slightly more resistant to tswv at  <dig> days post inoculation, but was less resistant to the virus at later times.
plants from line fkt4- <dig> were more resistant than wild-type control plants at all-time points except at  <dig> days after inoculation.
the resistance of fkt4- <dig> transgenic progeny over time is consistent with the results presented in table  <dig>  in which selfed plants from line fkt4- <dig> that contained the transgene were significantly more resistant to tswv than selfed plants from this line that did not contain the transgene.
response of transgenic petunia plants to tswv infection
the progeny of  <dig> transgenic petunia lines were tested for tswv resistance in a randomized complete block design.
in each of the replicates, wild-type non-transgenic controls grown under the same conditions were included among the transgenic lines.
three to four days after tswv inoculation, necrotic local lesions developed on inoculated leaves of test plants.
one transgenic line  had significantly smaller local lesion sizes and fewer numbers of local lesions compared with wild-type control plants .
twelve other transgenic lines were not significantly different from control plants in their levels of resistance to tswv infection as measured by the sizes and numbers of local lesions.
because the numbers of r <dig> petunia plants used in our experiments ranged from  <dig> to  <dig> for each line, all of the bioassayed plants were not screened by pcr for the presence of the transgene.
however, the data could still indicate a correlation of modified aox expression in the transgenic progeny with resistance to tswv infection, even though segregation of aox expression existed in the r <dig> population.
the average values of each replication were used for paired t-test of the progenies produced from transgenic petunia lines and wild-type control.
the p values in the table were estimated by the replications ranged from  <dig> to  <dig>
* indicates significant  difference.
CONCLUSIONS
our experiments demonstrate that transgenic tomato line fkt4- <dig> and transgenic petunia line fkp <dig>  both with elevated aox expression levels, have higher levels of resistance to tswv than control plants.
these results differ from the reported lack of resistance to tobacco mosaic virus  in transgenic tobacco with altered levels of aox  <cit> .
however, in these experiments with tobacco, only two transgenic lines were analyzed.
if more transgenic lines had been created and evaluated, different conclusions might have been reached.
several studies have shown that altered aox activity was positively correlated with resistance of transgenic tobacco and arabidopsis plants to tmv and cmv infection  <cit> .
other, contradictory results were found in tmv challenged tobacco and n. benthamiana  <cit> .
as more plant species and viruses have been used to elucidate antiviral mechanisms in plants, it has become clear that different host species can use different mechanisms to resist virus infection  <cit> .
our results support the hypothesis that the aox pathway may be associated in some way with plant resistance to viruses.
in our experiments and those of others, all plants with modified aox expression levels that have been evaluated have been challenged with only one virus.
it has not been reported how host species with altered aox levels respond to challenges by different plant viruses.
our transgenic tomato line fkt4- <dig> and petunia line fkp <dig> will be challenged with viruses other than tswv to evaluate their wide-spectrum virus resistance.

 mitochondrial alternative oxidases are important components of the alternative respiratory pathway of plants; aox genes have been isolated from several important plant species. synthesis of aox can be induced when the cytochrome pathway is inhibited, or when the plant is wounded, treated with ethylene, cycloheximide, chloramphenicol, or if the plant is exposed to cold environmental conditions. in addition, the aox pathway can also be induced by treatments with salicylic acid, nitric oxide, reactive oxygen species, high light intensities or pathogen challenge. because sa induction has been linked to the defense response in plants, it has been suggested that the alternative pathway might be associated with disease resistance in plants including resistance to viruses. evidence supporting this hypothesis includes the finding that elevated levels of aox in tobacco inhibit long-distance movement of cucumber mosaic virus and replication of tobacco mosaic virus and potato virus x. furthermore, additional works with cytochrome inhibitors and salicylhydroxamic acid have led to the proposal that the aox pathway and the products of the aox genes play a key role in the resistance of tobacco plants to virus infection. other studies have suggested, however, that aox is not a critical component of plant viral resistance but that it may play a role in the development of the hypersensitive response. elevated aox gene expression levels had no clear-cut effects on sa-induced resistance to systemic infection by tmv in transgenic tobacco. moreover, resistance to tmv in tobacco induced by antimycin a, an inhibitor of the cytochrome pathway, was repressed with increased alternative pathway capacity, and both sa- and aa-induced resistances were enhanced when alternative pathway capacity was reduced. furthermore, high-levels of alternative oxidase expression allowed increased tmv spread and the development of severe symptoms in nn-type tobacco and nicotiana benthamiana. the involvement of aox in virus resistance has been reported in a limited number of plant species and virus combinations, however, the mechanisms of this antiviral action varied. in order to accumulate more evidence that might further elucidate the association of aox with antiviral activity, we generated transgenic tomato and petunia lines with altered aox expression levels and evaluated their resistant levels to tomato spotted wilt virus.  tomato and petunia transformation and controlled pollination of transgenic lines tomato cultivar'healani' and petunia cultivar'sheer madness' were transformed with the leaox1au gene isolated from tomato to obtain transgenic lines. primers specific for the camv 35s promoter and 3'-end of the target inserts pbilaf and pbilac were used in pcr amplifications to detect the presence of integrated leaox1au sequences. both wild type and transgenic tomato plants contain the same alternative oxidase gene in their genomes, therefore the camv 35s promoter fragment dna, was used to probe ecori-digested genomic dna. a single ecor i recognition site is located near the left border of the inserted sequences, thus each band shown in figure should represent different insert locations. tomato lines cdt and ckt showed single bands, and may represent single copy transformants. the other lines showed to bands, indicating that these lines were transformed with multiple copies of the leaox1au construct. several putatively transgenic tomato lines were analyzed for aox rna expression by northern analyses using a leaox1-au specific orf dna fragment labeled with dig as a probe. leaox1-au was not detected in vector only or non-transgenic tomato plants. expression of aox could not be detected in lines cdt cdt cdt17- and cdt17- the remaining eleven transformed lines showed altered expression of leaox-1au ranging from to times the average signal intensity of the aox orf verses that of 18s rrna. no clear relationship between the aox copy number determined by southern analyses and the aox rna expression level determined by northern analyses was observed. the putative transgenic tomato lines were tested for their aox protein expression levels by western blot analyses. aox expression in non-transgenic and vector-only transformed tomato plants was below detection levels. thirty-two transgenic tomato lines showed increased expression of aox compared to aox expression levels in non-transgenic and vector-only transgenic tomato plants. however three transgenic lines did not show increased aox expression. based on pcr, southern, northern, and western blot analyses, a total of tomato and petunia lines were shown to be transformed with the leaox1au gene. the transgenic lines were self-pollinated and the harvested seeds were dried and stored at 4°c. seeds of tomato lines and petunia lines were germinated and grown in the greenhouse. when the tomato seedlings reached to cm in height, one leaf disk was collected from each plant using # cork borer. transgene constructs in the progeny were confirmed by pcr and southern hybridizations. only confirmed transgenic lines were analyzed further by tswv challenge. response of r generation transgenic tomato plants to tswv infection twenty to r1generation tomato plants from each line were evaluated for resistance to tswv. within these groups, to lines were confirmed to contain the transgene by pcr analyses. the results showed that within each line, all of the transgenic plants had lower elisa readings after tswv challenge than those of non-transgenic plants, indicating that the transgenic tomato plants had elevated resistance level to tswv infection. although no lines were found to be completely immune to tswv, paired t-tests indicated that the progeny from line fkt4- showed significantly lower elisa values than the wild-type control line. progeny from seven other transgenic lines did not have statistically significant enhanced resistance compared with wild-type controls, which might possibly be related to positional effects of the insertion event or if the transgene interacted somehow with other genes. * indicates significant difference response of transgenic tomato plants to tswv infection at different time points to evaluate the dynamic response to tswv infection, to plants of transgenic lines fkt fkt4- and wild-type controls were evaluated over time after virus challenge. tissues were sampled and elisa analyses were performed up to days after the second tswv inoculation. the data in figure are the average of three elisa values from 4- plants of each line. results show that plants of both transgenic lines and the control all developed tswv infections by days after challenge, which showed small dark spots, bronzed leaves that rolled upward, and dieback of young branches. line fkt was slightly more resistant to tswv at days post inoculation, but was less resistant to the virus at later times. plants from line fkt4- were more resistant than wild-type control plants at all-time points except at days after inoculation. the resistance of fkt4- transgenic progeny over time is consistent with the results presented in table in which selfed plants from line fkt4- that contained the transgene were significantly more resistant to tswv than selfed plants from this line that did not contain the transgene. response of transgenic petunia plants to tswv infection the progeny of transgenic petunia lines were tested for tswv resistance in a randomized complete block design. in each of the replicates, wild-type non-transgenic controls grown under the same conditions were included among the transgenic lines. three to four days after tswv inoculation, necrotic local lesions developed on inoculated leaves of test plants. one transgenic line had significantly smaller local lesion sizes and fewer numbers of local lesions compared with wild-type control plants. twelve other transgenic lines were not significantly different from control plants in their levels of resistance to tswv infection as measured by the sizes and numbers of local lesions. because the numbers of r petunia plants used in our experiments ranged from to for each line, all of the bioassayed plants were not screened by pcr for the presence of the transgene. however, the data could still indicate a correlation of modified aox expression in the transgenic progeny with resistance to tswv infection, even though segregation of aox expression existed in the r population. the average values of each replication were used for paired t-test of the progenies produced from transgenic petunia lines and wild-type control. the p values in the table were estimated by the replications ranged from to * indicates significant difference.  our experiments demonstrate that transgenic tomato line fkt4- and transgenic petunia line fkp both with elevated aox expression levels, have higher levels of resistance to tswv than control plants. these results differ from the reported lack of resistance to tobacco mosaic virus in transgenic tobacco with altered levels of aox. however, in these experiments with tobacco, only two transgenic lines were analyzed. if more transgenic lines had been created and evaluated, different conclusions might have been reached. several studies have shown that altered aox activity was positively correlated with resistance of transgenic tobacco and arabidopsis plants to tmv and cmv infection. other, contradictory results were found in tmv challenged tobacco and n. benthamiana. as more plant species and viruses have been used to elucidate antiviral mechanisms in plants, it has become clear that different host species can use different mechanisms to resist virus infection. our results support the hypothesis that the aox pathway may be associated in some way with plant resistance to viruses. in our experiments and those of others, all plants with modified aox expression levels that have been evaluated have been challenged with only one virus. it has not been reported how host species with altered aox levels respond to challenges by different plant viruses. our transgenic tomato line fkt4- and petunia line fkp will be challenged with viruses other than tswv to evaluate their wide-spectrum virus resistance. 
highly expressed aox transgenic tomato and petunia plants were selfed and transgenic r seedlings from tomato lines and petunia lines were used for bioassay. in all assayed transgenic tomato lines, a higher percentage of transgenic progeny had lower tswv levels than non-transgenic plants after challenge with tswv, and the significantly increased resistant levels of tomato and petunia lines identified in this study indicate that altered expression levels of aox in tomato and petunia can affect the levels of tswv resistance. we show here that the over-expression of the mitochondrial alternative oxidase in tomato and petunia is related to tswv resistance. tomato spotted wilt virus has a very wide host range, and is transmitted in a persistent manner by several species of thrips.
 tomato spotted wilt virus has a very wide host range, and is transmitted in a persistent manner by several species of thrips. these characteristics make this virus difficult to control. we show here that the over-expression of the mitochondrial alternative oxidase in tomato and petunia is related to tswv resistance.  the open reading frame and full-length sequence of the tomato aox gene leaox1au were cloned and introduced into tomato'healani' and petunia'sheer madness' using agrobacterium-mediated transformation. highly expressed aox transgenic tomato and petunia plants were selfed and transgenic r seedlings from tomato lines and petunia lines were used for bioassay. for each assayed line, to tomato r progeny in three replications and to petunia progeny in replications were challenged with tswv. enzyme-linked immunosorbent assays showed that the tswv levels in transgenic tomato line fkt4- was significantly lower than that of wild-type controls after challenge with tswv. in addition, transgenic petunia line fkp showed significantly less lesion number and smaller lesion size than non-transgenic controls after inoculation by tswv.  in all assayed transgenic tomato lines, a higher percentage of transgenic progeny had lower tswv levels than non-transgenic plants after challenge with tswv, and the significantly increased resistant levels of tomato and petunia lines identified in this study indicate that altered expression levels of aox in tomato and petunia can affect the levels of tswv resistance. 