Patent Application: US-201214345969-A

Abstract:
disease in food crops caused by fungal pathogens is a major concern to the agricultural industry , with annual losses typically in the billions of dollars . fusarium graminearum , also known as gibberella zeae , is known to cause , among other diseases , headblight disease in wheat and stalk and ear rot in maize . disease caused by fusarium graminearum has proven to be a difficult disease to manage because of limitations of control options . disclosed herein are nucleic acid sequences which have been proven to provide corn and soybean with resistance to fusarium graminearum . also disclosed herein are methods of using the nucleic acid sequences , and plants comprising the nucleic acid sequences .

Description:
this invention relates to nucleic acid sequences , preferably isolated nucleic acid sequences , which confer resistance to fungal disease upon host plants . this invention is also drawn to plants expressing the nucleic acid sequences , whereby the plants are resistant to fungal disease . these plants which express these nucleic acid sequences are useful in controlling fungal disease caused by a pathogenic fungus , particularly a fusarium species , and more particularly fusarium graminearum . in one embodiment , the present invention comprises a single - stranded nucleic acid molecule , or an isolated single - stranded nucleic acid molecule , comprising a first sequence and a second sequence , wherein the first sequence comprises a sequence obtained from a gene that encodes a fungal ribosomal rna , and the second sequence comprises a sequence capable of forming a duplex with the first sequence . in another embodiment , the fungal ribosomal rna is from a fungus in the genus fusarium . in another embodiment , the fungal ribosomal rna is the 28s ribosome from fusarium graminearum . in another embodiment , the first sequence is selected from the group consisting of seq id nos : 1 , 3 , 5 , and 7 . in another embodiment , the second sequence is selected from the group consisting of seq id nos : 2 , 4 , 6 , and 8 . in another embodiment , the single - stranded nucleic acid molecule further comprises a backbone sequence between the first sequence and the second sequence . in another embodiment , the backbone sequence comprises at least nucleotides 41 to 167 of seq id no : 12 . in another embodiment , the single - stranded nucleic acid sequence is capable of forming a hairpin . in another embodiment , the single - stranded nucleic acid molecule is synthetic . in another embodiment , the nucleic acid is rna or dna or an dna / rna hybrid . in yet another embodiment , the single - stranded nuckeic acid molecule of the invention is active against a fusarium fungus or a phakopsora fungus . in another embodiment , the fusarium fungus is fusarium graminearum . in another embodiment , the phakopsora fungus is phakopsora pachyrhizi . in another embodiment , the present invention comprises an expression cassette comprising at least a first nucleic acid sequence which encodes for a first single - stranded nucleic acid molecule comprising a first sequence and a second sequence , wherein the first sequence comprises a sequence obtained from a gene that encodes a fungal ribosomal rna , and the second sequence comprises a sequence capable of forming a duplex with the first sequence . in another embodiment , the expression cassette further comprises a second nucleic acid sequence , wherein the first single - stranded molecule and the second single - stranded molecule do no comprise identical first sequences . in another embodiment , the first single - stranded molecule comprises a first sequence selected from the group consisting of seq id nos : 1 , 3 , 5 , and 7 , and the second single - stranded molecule comprises a first sequence different from the first sequence in the first single - stranded molecule . in another embodiment , the first single - stranded molecule comprises a first sequence comprising seq id no : 1 , and the second single - stranded molecule comprises a first sequence comprising seq id no : 3 . in another embodiment , the expression cassette comprises seq id no : 13 . in another embodiment , the present invention comprises a vector comprising an expression cassette comprising at least a first nucleic acid sequence which encodes for a first single - stranded nucleic acid molecule comprising a first sequence and a second sequence , wherein the first sequence comprises a sequence obtained from a gene that encodes a fungal ribosomal rna , and the second sequence comprises a sequence capable of forming a duplex with the first sequence . in another embodiment , the vector comprises seq id no : 14 or 15 . in another embodiment , the present invention comprises a non - human host cell comprising an expression cassette comprising at least a first nucleic acid sequence which encodes for a first single - stranded nucleic acid molecule comprising a first sequence and a second sequence , wherein the first sequence comprises a sequence obtained from a fungal ribosome , and the second sequence comprises a sequence capable of forming a duplex with the first sequence . in another embodiment , the non - human host cell is selected from the group consisting of bacteria , virus , fungus , plant , and animal cells . in another embodiment , the non - human host cell is a plant cell . in another embodiment , the present invention comprises a plant comprising a plant cell comprising an expression cassette comprising at least a first nucleic acid sequence which encodes for a first single - stranded nucleic acid molecule comprising a first sequence and a second sequence , wherein the first sequence comprises a sequence obtained from a gene that encodes a fungal ribosomal rna and the second sequence comprises a sequence capable of forming a duplex with the first sequence . in another embodiment , the plant is a monocot . in another embodiment , the monocot is maize . in another embodiment , the plant is a dicot . in another embodiment , the dicot is soybean . in yet another embodiment , the transgenic plant of the invention is resistant to a fusarium fungus or a phakopsora fungus . in another embodiment , the fusarium fungus is fusarium graminearum . in another embodiment , the phakopsora fungus is phakopsora pachyrhizi . in another embodiment , the present invention comprises a method of producing a plant resistant to a fungal pathogen , comprising the steps of : ( a ) obtaining an expression cassette comprising a nucleotide sequence encoding a single - stranded nucleic acid molecule , or an isolated single - stranded nucleic acid molecule , comprising a first sequence and a second sequence , wherein the first sequence comprises a sequence obtained from a gene that encodes a fungal ribosomal rna , and the second sequence comprises a sequence capable of forming a duplex with the first sequence ; ( b ) inserting the expression cassette into the genome of a plant cell ; and ( c ) generating a plant from the plant cell ; wherein the plant is resistant to a fungal pathogen . in another embodiment , the isolated single - stranded nucleic acid molecule comprises a first sequence selected from the group consisting of seq id nos : 1 , 3 , 5 , and 7 . in another embodiment , the plant cell is a maize plant cell . in another embodiment , the plant is a maize plant . in another embodiment , the plant cell is a soybean plant cell . in another embodiment , the plant is a soybean plant . in another embodiment , a method of the inventionprodecues a plant that is resistant to a fusarium fungus or a phakopsora fungus . in another embodiment , the fusarium fungus is fusarium graminearum . in another embodiment , the phakopsora fungus is phakopsora pachyrhizi . all the features disclosed in this specification ( including any accompanying claims , abstract , and drawings ) may be replaced by alternative features serving the same , equivalent or similar purpose , unless expressly stated otherwise . thus , unless expressly stated otherwise , each feature disclosed is one example only of a generic series of equivalent or similar features . these embodiments are better understood in light of the examples provided below . potential micro rna ( mirna ) targets were identified from scans of genomic dna encoding the 28s ribosomal rna ( rrna ) derived from the maize fungal pathogen fusarium graminearum ( also known as gibberella zeae ). the genomic dna sequence encoding 28s ribosomal rna from fusarium graminearum harbors a 125 bp sequence that when expressed as an rna duplex exhibits high in vitro anti - fungal activity as measured by inhibition of spore germination . seq id no : 11 shows a partial sequence of the fusarium graminearum 28s ribosomal rna gene ( locus : ay188924 ). the sequence from nucleotides 476 - 600 has in vitro anti - fungal activity as an rna duplex ). four passenger mirna sequences ( seq id nos : 18 , 19 , 5 , and 7 ), and their antisense guide sequences ( seq id nos : 2 , 4 , 6 , and 8 ) were identified ( table 1 ) based on the partial sequence encoding the 28s ribosomal rna and selected for further testing . in each of the sequences in table 1 , a double nucleotide overhang , herein represented as “ nn ”, is included on the 3 ′ end . the overhang is needed for dicer to process the duplex . as used herein , “ n ” is meant to represent any nucleotide . therefore , any nucleotide , or any combination of nucleotides , can be used in the overhang . in one aspect , any combination of a , t , u , g , or c is used in the overhang . in another aspect , the same nucleotide is used twice in the overhang . in another aspect , the overhang is tt or uu . the antisense guide strands are responsible for driving the rna degradation mechanism within the plant . novel synthetic rna duplexes comprising at least two passenger sequences selected from the group consisting of seq id nos : 1 - 4 were created and tested by in vitro bioassays against f . graminearum and the soybean rust pathogen ( phakopsora pachyrhizi ). synthetic rna duplexes were created and tested by in vitro bioassays . these assays are described in u . s . patent application publication no . 2010 / 0257634 a1 ( ser . no . 12 / 753 , 901 ), incorporated herein by reference in its entirety . approximately 10 μg of these individual rna duplexes were incubated with spores of the soybean rust fungus ( phakopsora pachyrhizi ) then assessed for anti - fungal activity as measured by percent inhibition of germination and appressorium formation . data shown in table 2 indicate that rna duplex fgrna - 1 ( comprising seq id nos : 18 & amp ; 2 ) and rna duplex fgrna - 2 ( comprising seq id nos : 19 & amp ; 4 ) rate the highest level of inhibition . rna duplex fgrna - 3 ( comprising seq id nos : 5 & amp ; 6 ) and rna duplex fgrna - 4 ( comprising seq id nos : 7 & amp ; 8 ) have a moderate level of inhibitory activity . importantly , the negative control ( fgrna - 5 ), which comprises nonsense rna sequences seq id nos : 9 & amp ; 10 , had virtually no affect on spore germination or appressorium formation . based on these data , synthetic mirna in planta expression cassettes were created based on the endogenous soybean micro - rna mir319 , seq id no : 12 ( subramanian , et al . 2008 ). being the better performing duplexes , fgrna - 1 and fgrna - 2 were chosen for further development . the passenger strands for fgrna - 1 and fgrna - 2 were engineered so that each would mimic the folding and mismatches that mir319 possesses when folded . therefore , seq id no : 18 was engineered to become seq id no : 1 , and seq id no : 19 was engineered to become seq id no : 3 . the passenger and guide strand sequences of mir319 ( nucleotides 21 - 40 for passenger and 169 - 188 for guide of seq id no : 12 ) were replaced by those sequences derived from fgrna - 1 ( comprising seq id nos : 1 & amp ; 2 ). the passenger and guide strand sequences of mir319 ( nucleotides 21 - 40 for passenger and 169 - 188 for guide of seq id no : 12 ) were replaced by those sequences derived from fgrna - 2 ( comprising seq id nos : 3 & amp ; 4 ). see fig2 , which shows the folded stem - loop of mir319 ( fig2 a ), fgrna - 2 ( fig2 b ), and fgrna - 1 ( fig2 c ). these mir319 derived expression elements were linked in a novel tandem dual - expression array to a cestrum viral promoter and a nos terminator ( fig1 ). subsequently , the plant expression cassette was ligated to binary vectors for soybean or maize transformation ( seq id nos : 14 and 15 , respectively ). these synthetic micro rnas stem - loop structures are then processed by the plants endogenous dcl1 - hyl1 - se protein complex ( dong , et al . 2008 . pnas 105 ( 29 ): 9970 - 9975 ) to produce the anti - fungal mirnas . these bioassays confirmed the ability of these rna duplexes to prevent germination of fungal spores . to express these anti - fungal duplexes in planta , novel mirna gene expression cassettes were created for transformation of maize and soybean . the passenger and guide sequences of the soybean endogenous mir319 stem - loop were modified , stacked in a duplex and used to create maize and soybean transformation vectors , as shown above . maize and soybean transformation experiments were initiated with mannose or hygromycin selection , respectively . the t o - generation events were analyzed by qrt - pcr assays for the presence of guide strand mirnas derived from fgrna - 1 and fgrna - 2 . the qrt - pct assay is sensitive and accurate for determining transcript levels of rna . briefly , rna is purified from tissue samples and the target sequence is reverse transcribed into a dna molecule . a reference rna molecule ( usually of a constitutively expressed gene , such as elongation factor efla ) is also reverse transcribed for control purposes . the dna molecules for the target sequence and the reference sequence are then amplified using real - time pcr . relative expression levels are determined by comparing the cycle threshold ( ct ) of the target sequence and the reference sequence . results from the qrt - pcr analysis proved that both mirnas are in fact expressed in both t 0 - generation soybean and corn events ( table 3 ). the analysis of t 0 - generation maize and soybean events by qrt - pcr confirmed the expression of specific anti - fungal guide strand sequences . this is the first demonstration of cross - species expression of synthetic mirna in maize and soybean . further , this proves that mirna can be expressed in planta by a single expression cassette as a duplex and that a dual - tandem array in a single expression cassette can be processed correctly . interestingly , the stem - loop comprising fgrna - 2 , which is closer to the promoter , is detected at a much higher level than fgrna - 1 . these events listed in table 3 were self - pollinated to create the t 1 - generation of seed for further testing . unfortunately , the single soybean event was a chimeric plant , and therefore , the trait was not inherited in the next generation . however , ten of the maize events were successfully selfed . the t 1 - generation plants were sampled for zygosity analysis followed by qrt - pcr . the analysis of the t 1 - generation plants , summarized in table 4 , found those plants , whether homozygous or heterozygous , derived from independent events expressed the anti - fungal mirnas . as expected , the null - siblings were negative for fgrna - 1 and fgrna - 2 expression as determined by qrt - pcr . consistent with the t 0 generation analysis , the fgrna - 2 which is closer to the promoter is expressed at a much higher level than fgrna - 1 in the t 1 - generation analysis . greenhouse experiments confirmed expression of these mirna in t 1 - generation maize lines . molecular characterization identified homozygous , heterozygous and null - sibling maize plants . these plants were used in detached leaf bioassays by inoculation with spores of f . graminearum . at 10 days post - inoculation , leaves were rated for disease severity . results show improved tolerance to f . graminearum on leaves taken from homozygous or heterozygous plants compared to either null - sibs or non - transgenic maize leaves . the fgrna - 1 and fgrna - 2 guide strands were originally identified by in vitro bioassays for their ability to prevent f . graminearum spore germination at a rate of & gt ; 90 % efficacy ( table 5 .). subsequently , detached leaf bioassays were performed on t 1 - generation ( homozygous or heterozygous ) that tested positive by qrt - pcr . leaves were collected , placed in a humidity chamber followed by inoculation with f . graminearum spores ( 25 , 000 spores / ml ). leaves from null - siblings or non - transgenic maize served as negative controls . a disease severity rating was used to rate each of the individual leaves tested in these experiments 6 - 10 days post inoculation ( 0 = no disease , 1 = trace , 2 = low , 3 = intermediate , and 4 = severe ). see table 6 . the null - siblings or nontransgenic leaves showed intermediate or severe levels of disease , while most leaves expressing fgrna - 1 and fgrna - 2 showed no signs of disease . in a second experiment , leaves from these same events were inoculated with the maize anthracnose pathogen colletotrichum graminicola ( anamorph glomerella graminicola ) at 25 , 000 spores / ml . none of these events expressing fgrna - 1 and fgrna - 2 have tolerance to this fungal pathogen when rated 6 - 10 days post inoculation . this experiment demonstrates the specificity of the anti - fungal mirnas targeting f . graminearum . secondly , the positive results observed are most likely not the result of the activation of endogenous maize disease resistance mechanisms . the t 1 - generation homozygous and null - siblings from independent maize events comprising seq id no : 13 were selfed to increase t 2 seed . ragdoll bioassays were performed on the t 2 seed . a ragdoll bioassay test consisted of 10 seeds spaced in a line 10 cm from the top edge of a layer of 31 cm by 61 cm germination paper and moistened with distilled , deionized h 2 o . a spore suspension of consisting of macroconidia of fusarium graminearum , quantified to 1 × 10 6 spores / ml , is dropped onto each seed with a dropper . a second pre - moistened sheet of germination paper was placed over the first layer , and the entire assembly was rolled along the short axis and secured with rubber bands . units were placed in a plastic bag and incubated vertically in an incubator for approximately 72 hours at 12 ° c . ( assay parameters were adjusted to give maximum root discoloration without killing the plants , therefore , time in the incubator varied ; too much disease meant less time in the 12 ° c . incubator ), then moved to another incubator for 96 hours at 25 ° c . in the light ( for 16 hour intervals ) and 20 ° c . in the dark ( 8 hour intervals ). at the end of the incubation period , bioassay units were unrolled , and root discoloration and germination rates were recorded in table 7 , below . control maize seeds from hybrid lines 09mz000080 and 09mz000084 were included in ragdolls 15 - 18 . the control seeds performed better due to the fact that they represent a hybrid genetic background , whereas the test t 2 seeds ( in ragdolls 1 - 14 ) represent inbred lines which were expected to perform poorly . it is submitted that the poor results are due to the lines being inbred and not to the performance of fgrna - 1 and fgrna - 2 stem - loops . this is supported by the generally poor germination rate , as observed during the ragdoll bioassays . additionally problematic is the contamination by penicillium in ragdolls 3 and 4 , and tricoderma in ragdoll 11 . t 2 seeds comprising seq id no : 13 are backcrossed into a hybrid maize genetic background . these seeds display an increased resistance to disease caused by fusarium graminearum due to the expression of fgrna - 1 and fgrna - 2 stem - loops . a second round of soybean transformation experimnets were carried out using the fgrna - 1 and fgrna - 2 mirna molecules described above . t o - generation events were analyzed for the presence of guide strand mirnas derived from fgrna - 1 and fgrna - 2 as described above . relative expression levels of the specific guide strands were comparable to the levels disclosed in table 3 . positive t 0 events were self pollinated to create the t 1 - generation of seed . plants grown from t 1 seed were sampled for zygosity analysis followed qrt - pcr as cdescribed above . eight of the highest expressing events were selected for generation of t 2 seed and for testing against soybean fungal diseases . the transgenic t2 soybean plants expressing the fgrna - 1 and fgrna - 2 mirna molecules were evaluated for resistance to the fungus phakopsora pachyrhizi , the causative agent of soybean rust disease . soybean rust spores were collected from inoculated leaves of non - transgenic susceptible soybean variety “ jack ” by washing leaves in water plus 0 . 01 % tween 20 . the spore concentration was adjusted to about 500 , 000 spores per ml . plants from the transgenic soybean lines expressing fgrna - 1 and fgrna - 2 were inoculated at the v - 2 stage . at about 10 - 14 days post inoculation , the first trifoliate leaf was rated ( scale 0 - 100 %) for disease severity . three separate whole - plant trials were carried out in a greenhouse . in the first experiment , some of the events showed a decrease in disease severity . however , the incidence of disease was low in all treatments including the susceptible control soybean line ( jack ), i . e . there was not adequate spore germination to have a conclusive test . data from two further trials showed that some plants in all the transgenic events had reduced disease compared to the non - transgenic control ( soybean variety jack ). in addition , in the second trial all plants from two events ( 4b001a104 and 11b004a218 ) had reduced disease and in the third trial all plants from three events ( 4b001a104 , 11b004a218 and 6b001a149 ) had reduced levels of disease compared to the non - transgenic control ( soybean variety jack ). the results of trials 2 and 3 are shown in table 8 . the “% disease ” column indicates the percent of leaf area infected . the “% control ” column indicates the reduction in disease compared to the susceptible control . these results clearly show fgrna - 1 and fgrna - 2 , which were designed to target 28s ribosomal rna in fusarium graminearum , also target the 28s ribosomal rna in phakopsora pachyrhizi . thus , such rnai molecules have utility in multiple crops to target and control multiple diseases . such rnai molecules are particularly useful in controlling a fusarium fungus , for example fusarium graminearum , or a phakopsora fungus , for example phakopsora pachyrhizi . all publications and patent applications are herein incorporated by reference to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference . although the foregoing invention has been described in some detail by way of illustration and example for purposes of clarity of understanding , it will be obvious that certain changes and modifications may be practiced within the scope of the list of the foregoing embodiments and the appended claims . 1 . mcmullen , m ., jones , r ., and gallenberg , d . 1997 . scab of wheat and barley : a re - 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