Patent Application: US-201113809062-A

Abstract:
the present invention relates to plants having increased pre - formed defense , their production and uses . the invention more particularly discloses plants which overexpress a p33kd or burp protein , or an ortholog thereof , and exhibit an increased pre - formed resistance to pathogens .

Description:
partial resistance to plant pathogens is extensively used in breeding programs since it could contribute to resistance durability . partial resistance often builds up during plant development and confers quantitative and usually broad - spectrum resistance . however , very little is known on the mechanisms underlying partial resistance . partial resistance is often explained by poorly effective induction of plant defense systems . by exploring rice natural diversity , we asked whether expression of defense systems before infection could explain partial resistance towards the major fungal pathogen magnaporthe oryzae . the constitutive expression of 21 defense - related genes belonging to the defense system was monitored in 23 randomly sampled plants for which partial resistance was measured . we identified a strong correlation between the expression of p33 kd or burp before infection and partial resistance . increasing constitutive expression of these genes also correlated with the establishment of partial resistance during plant development . these results indicate that constitutive expression of p33 kd or burp can cause , stimulate or maintain resistance in plants . the finding of this preformed defense system allows development of new plants having improved properties . the term “ preformed resistance ” designates a resistance caused or stimulated by gene expression prior to infection by a pathogen . this resistance is a priori not a gene - to - gene resistance mechanism , by which a cell gene product is produced in response to and / or interacts with a pathogen gene product . preformed resistance thus prevents plants from being infected and has a broad spectrum against different types of pathogens . the term “ p33 kd ” or “ 33 kda ” designates the 33 kd secretory protein having accession number os03g16950 , as well as any natural variants thereof and its orthologs in plants . this protein has been sequenced and isolated . a sequence of the rice protein is provided below ( seq id no : 2 ): the term “ burp ” designates the burp protein or domain having accession number os05g12640 , as well as any natural variants thereof and its orthologs in plants . this protein has been sequenced and isolated . within the context of the present invention , the term p33 kd or burp “ gene ” designates any nucleic acid that codes for a p33 kd or burp protein . an exemplary gene encoding p33 kd comprises the nucleic acid represented below ( seq id no : 1 ), which corresponds to the rice gene sequence : a further example of a p33 kd gene is a nucleic acid sequence comprising seq id no : 3 , which has been artificially created and encodes p33 kd . within the context of the present invention , the term “ ortholog ” designates a related gene or protein from a distinct species , having a level of sequence identity to a reference protein above 50 % and a similar activity . an ortholog of p33 kd or burp is most preferably a gene or protein from a distinct species having a common ancestor with p33 kd or burp , having a similar activity , and having a degree of sequence identity with superior to 50 %, more preferably at least 60 %, especially preferably at least 65 , 75 , 80 , 90 , 95 % or more sequence identity . alignment between two sequences and their degree of identity are defined by comparison with the entire polypeptide sequences with the software “ needle ” ( needleman et wunsch , j . mol . biol ., 48 , 443 - 453 , 1970 ) with the parameters by default : & lt ;& lt ; matrix & gt ;≦: eblosum62 , & lt ;& lt ; gap penalty & gt ;& gt ;: 10 . 0 et & lt ;& lt ; extend penalty & gt ;& gt ;: 0 . 5 . specific examples of p33 kd ortholog sequences have been identified by the inventors and are listed below : in a preferred embodiment , the invention relates to constructs and plants containing or expressing a p33 kd protein comprising seq id no : 2 or a sequence having a degree of sequence identity with seq id no : 2 superior to 65 %. within the context of the present invention , the term “ pathogens ” designates all pathogens of plants in general . more preferably the pathogens are fungal pathogens . in a particular embodiment , fungal pathogens are cereal fungal pathogens . examples of such pathogens include , without limitation , magnaporthe , puccinia , aspergillus , ustilago , rhizoctonia , septoria , erisyphe and fusarium species . in the most preferred embodiment , the pathogen is magnaporthe oryzae . the invention is particularly suited to create rice resistant to magnaporthe . a promoter is “ heterologous ” to a gene when said promoter is not naturally associated to said gene . a heterologous promoter may be natural , synthetic , recombinant , hybrid , etc . it may be of cellular or viral origin . the heterologous promoter is preferably a strong and / or constitutive promoter . constitutive promoters may include , for example camv 35s promoter ( odell et al . ( 1985 ) nature 313 , 9810 - 812 ), rice actin promoter ( mcelroy et al . ( 1990 ) plant cell 2 : 163 - 171 ) and ubiquitin promoter ( christian et al . ( 1989 ) plant mol . biol . 18 ( 675 - 689 ); pemu ( last et al . ( 1991 ) theor appl . genet . 81 : 581 - 588 ); mas ( velten et al . ( 1984 ) embo j . 3 . 2723 - 2730 ); als promoter ( u . s . application ser . no . 08 / 409 , 297 ), and the like . non - limitative examples of constitutive promoters that are commonly used are the cauliflower mosaic virus ( camv ) 35s promoter , the nopaline synthase ( nos ) promoter , the cassava vein mosaic virus ( csvmv ) promoter ( verdaguer et al ., 1996 ), the rice actin promoter followed by the rice actin intron ( rap - rai ) contained in the plasmid pact1 - f4 ( mcelroy et al ., 1991 ), the corn ubiquitin or rice ubiquitin 3 promoter ( sivamani et qu , plant mol biol ., 60 : 225 - 239 , 2006 ). non - limitative examples of organ or tissue specific promoters that can be used in the present invention include for instance root specific promoter like ppro110 from rice , the kernel specific promoter high molecular weight ( hmw ) promoter ( thomas and flavell , 1990 ), or the leaf specific promoters as ppepc promoter ( jeanneau et al ., 2002 ), or the rubisco small subunit promoter ( rbcs ) ( katayama et al ., 2000 ) which is specific of the bundle - sheath . inducible promoters include for instance pathogene responsive or drought stress responsive promoters , such as the rd29a promoter which comprises a dehydration - responsive element ( kasuga et al ., 1999 ; narusaka et al ., 2003 ), or the senescence specific sag12 promoter ( noh and amasino , 1999 ). within the context of this invention , the term “ overexpressed ” or “ overexpression ” indicates either an increase in the level of active protein present in the cell or plant and / or a modified expression profile of a protein , such as a constitutive expression . overexpression may be obtained by techniques known per se in the art such as , without limitation , by genetic means , enzymatic techniques , chemical methods , or combinations thereof . overexpression may be conducted at the level of dna , mrna or protein , and induce the expression ( e . g ., transcription or translation ) or the activity of the protein . a preferred overexpression method affects expression and leads to the increased production of a functional protein in the cells . it should be noted that the induction may be transient or permanent . preferably , an overexpression designates an increase by at least 10 %, preferably at least 20 %, more preferably at least 30 % or even more , as compared to reference expression or to an average value . in a first embodiment , overexpression is induced by a mutation in the coding gene , for example point mutation , deletion , insertion and / or substitution of one or more nucleotides in a dna sequence . this may be performed by techniques known per se in the art , such as e . g ., site - specific mutagenesis , ethyl methanesulfonate ( ems ) mutagenesis , targeting induced local lesions in genomes ( tilling ), homologous recombination , conjugation , etc . a particular approach is gene overexpression by insertion a dna sequence through transposon mutagenesis using mobile genetic elements called transposons , which may be of natural or artificial origin . mutations may also be introduced within the sequence of the promoter . mutations in the coding sequence may result in gain of function by increasing biological activity and efficacy of the protein . alternatively , introducing mutations into the promoter sequence may result in gain of function by induction of the promoter activity by controlling and enhancing transcription of the gene . in a particular embodiment , overexpression is induced by introduction into a plant of an expression cassette comprising a nucleic acid sequence coding for the protein under control of a promoter enabling the expression of said nucleic acid sequence . overexpression may also be performed transiently , e . g ., by applying ( e . g ., spraying ) an exogenous agent to the plant , for example molecules that induce the protein expression or activity . a preferred overexpression is a constitutive expression under control of a constitutive promoter . a nucleic acid molecule may be introduced into a plant cell by any means , including transfection , transformation , transduction , electroporation , particle bombardment , agroinfection , etc . in a preferred embodiment , a nucleic acid molecule is introduced via agrobacterium transformation using the ti plasmid as described e . g ., by toki et al . ( 2006 ). a variety of techniques for genetic transformation of plant cells or plants are available in the art . by way of non - limitative examples , one can mention methods of direct transfer of genes such as direct micro - injection into plant embryoids , vacuum infiltration ( bechtold et al . 1993 ) or electroporation ( chupeau et al ., 1989 ) or the bombardment by gun of particules covered with the plasmidic dna of interest ( fromm et al ., 1990 ; finer et al ., 1992 ). agrobacterium mediated transformation methods may also be used such as agrobacterium tumefaciens , in particular according to the method described in the article by an et al . ( 1986 ), or agrobacterium rhizogenes , in particular according to the method described in the article by guerche et al ., ( 1987 ). according to a particular embodiment , it is possible to use the method described by ishida et al . ( 1996 ) for the transformation of maize . according to another embodiment , the wheat is transformed according to the method described in international application wo 00 / 63398 . according to the present invention , the introduced nucleic acid molecule may be maintained in the plant cell stably . alternatively , the introduced nucleic acid molecule may be transiently expressed or transiently active . selection of a plant which overexpress a p33 kd or burp protein can be made by techniques known per se to the skilled person ( e . g ., pcr , hybridization , use of a selectable marker gene , protein dosing , western blot , etc .). plant generation from the modified cells can be obtained using methods known per se to the skilled worker . in particular , it is possible to induce , from callus cultures or other undifferentiated cell biomasses , the formation of shoots and roots . the plantlets thus obtained can be planted out and used for cultivation . methods for regenerating plants from cells are described , for example , by fennell et al . ( 1992 ) plant cell rep . 11 : 567 - 570 ; stoeger et al ( 1995 ) plant cell rep . 14 : 273 - 278 ; jahne et al . ( 1994 ) theor . appl . genet . 89 : 525 - 533 . the resulting plants can be bred and hybridized according to techniques known in the art . preferably , two or more generations should be grown in order to ensure that the genotype or phenotype is stable and hereditary . selection of plants having an increased resistance to a pathogen can be done by applying the pathogen to the plant , determining resistance and comparing to a wild type plant . within the context of this invention , the term “ increased ” resistance to pathogen means a resistance superior to that of a control plant such as a wild type plant , to which the method of the invention has not been applied . the “ increased ” resistance also designates a reduced , weakened or prevented manifestation of the disease symptoms provoked by a pathogen . the terms “ wheat plant ” and “ wheat plant cell ” as used herein , include any plant of plant cell of the genus triticum , preferably of the species triticum aestivum l . ( bread wheat ). the invention also comprises host cells containing a recombinant dna construct of the invention . these host cells can be prokaryotic cells or eukaryotic cells , in particular plant cells , and preferably maize cells . the invention also comprises wheat plants genetically transformed by a recombinant dna construct of the invention , and overexpressing a p33 kd or burp or an ortholog as defined above . in said transgenic plants a dna construct of the invention is comprised in a transgene stably integrated in the plant genome , so that it is passed onto successive plant generations . thus the transgenic wheat plants of the invention include not only the plants resulting from the initial transgenesis , but also their descendants , as far as they contain a recombinant dna construct of the invention . the overexpression of a p33 kd or burp as defined above in said plants provides them an improved grain filling , when compared with a plant devoid of said transgene ( s ). the invention also comprises a transgenic wheat plant , obtainable by a method of the invention , overexpressing a p33 kd or burp as defined above . the invention further comprises a transgenic wheat plant or an isolated organ or tissue thereof comprising , stably integrated in its genome , a recombinant expression cassette comprising a polynucleotide encoding a p33 kd or burp as defined above . accordingly , the invention also encompasses isolated organs or tissues of said transgenic wheat plant ( such as seeds , leafs , flowers , roots , stems , ears ) containing a recombinant expression cassette of the invention . further aspects and advantages of the invention will be disclosed in the following experimental section , which should be considered as illustrative . rice diversity was estimated from garris et al [ 53 ]. the names used for the rice accessions are the names used for the mini germplasm bank . seeds were obtained from cirad - center for biological resource ( france ). rice was grown as in [ 11 ]. three types of genes along the disease resistance pathway were selected : one prr , 12 regulators and 12 defense genes ( fig1 ). this classification of genes was sometimes arbitrary as for some genes the putative function was unknown ( e . g . 33 kda secretory protein ). the role of some putative regulator genes in rice was deduced from gene expression studies ( e . g . the eds5 gene ) [ 11 ] and by transcriptome information gathered in the archipelago database [ 54 , 12 ]. the npr1 [ 8 ], rci1 [ 36 ] and ein2 [ 35 ] genes were included as markers for the salicylic acid , the jasmonic acid and the ethylene pathways respectively . other genes were included in this study as regulator genes ( hlhdb , znfg1 , znfg2 and znpgxs ) given their annotations and expression studies ( fig1 ). defense genes were genes for which the annotation and expression studies suggest a direct role in limiting pathogen growth . for example the chi gene potentially degrades chitin , the major component of fungal cell - wall . altogether , these genes are representative of the defense arsenal . all genes used in this work were , to some extent , differentially expressed upon infection ( fig1 ). twenty - eight cultivars were characterized for partial resistance ( fig1 ). plants were grown and inoculated when 3 - weeks old with spore suspensions of 50000 spores / ml as in [ 11 ]. the quantity of fungal mass for four isolates of m . oryzae ( cd101 , cd203 , cl26 , cm28 ) was measured by q - pcr on dna extracted 7 days post - inoculation , in three independent experiments ( 8 leaves / experiment ). fungal growth was estimated using taqman ® technology with the maggy transposon for m . oryzae ( maggy taqman probe tgagcagccaacgccgccacaa ) and the actin gene for rice ( actin taqman probe atcacgcccagcaaggtcgagacg ). primers are given in fig2 . the eurogentec taqman kit was used on a stratagene mx300p qpcr machine . in addition to classical symptoms ( data not shown ), a total of 12 values ( 4 isolates x 3 biological replicates ) were used to build the partial resistance index . the inverse of the mean of the 12 measures obtained per cultivar was assumed to be an estimation of partial resistance ( fig1 ). rnas were extracted and gene expression measured as in [ 11 ]. all expression experiments were done two to three times in biologically independent experiments . the primers used are listed in fig2 . calculation of gene expression was normalized using the rice actin gene and expression formula from pfaffl [ 56 ]. although naturally occurring dna polymorphism only slightly modifies gene expression using oligo - nucleotide microarrays [ 29 ], qrt - pcr that involves longer dna sequences could be sensitive to dna polymorphism . thus we evaluated the variability of qrt - pcr measures for eight genes in six representative rice accessions ( fig1 ). four genes ( 33 kda , burp , chi and hsp90 ) showed qrt - pcr efficiencies more variable than in the actin control but the overall variation was low (& lt ; 20 %). this variability was not related to indica / japonica sub - group or elevated / low partial resistance classes . the four other genes tested ( pbz1 , hlhdb , spl7 and znfg2 ) showed very limited variability across rice diversity . we concluded that the qrt - pcr conditions used in this study , although influenced by dna polymorphism , were sufficient to evaluate expression variability across rice diversity . the pearson correlation coefficient value and the test of the value being different from zero were estimated with functions “ cor ” and “ cor . test ” in r stats package ( http :// www . r - project . org ). for principal component analysis ( pca ), the numeric variables were log 2 transformed . pca were done with function “ dudi . pca ” in ade4 library ( http :// pbil . univ - lyon1 . fr / ade - 4 ) for r ( http :// www . r - project . org ). for anova , the variables were log 2 transformed . the anova models “ m . oryzae quantity = gene 1 expression value + gene 2 expression value + . . . + gene x expression value ” were tested with the “ lm ” function in r stats package . models were validated with shapiro . test function ( residues normality ) in r stats package and hmctest or bptest functions ( heteroskedasticity ) in lmtest library ( http :// cran . r - project . org / web / packages / lmtest / index . html ) for r . models explanation was done with the anova function of r stats package . for sa , frozen ( liquid nitrogen ) leaf tissues ( about 0 . 5 g ) were ground in 0 . 5 ml of 90 % methanol . [ 14 c ] sa was then added ( 60 μl ) as tracer to each tube . after centrifugation ( 15 min , 16000g ), the residue was extracted again with 100 % methanol ( 0 . 5 ml ) and , after centrifugation ( 15 min , 16000 g ), the second supernatant was added to the first one . after a third centrifugation ( 10 min , 16000 g ) combined supernatants were evaporated to dryness with a speedvac ( 5 h , 30 ° c .). for each sample , the dried extract was resuspended in hot water ( 80 ° c ., 0 . 4 ml ) and hcl 12 n ( 0 . 2 ml ) and incubated for 45 min at 80 ° c . in a water bath . after cooling , 1 ml of ether was added and after centrifugation , the organic phase was collected . a new step of phase partitioning was achieved on the aqueous phase . the two organic phases were then added and evaporated to dryness under nitrogen flux . final samples were resuspended in 200 μl of injection buffer ( 10 % acetonitrile , 90 % sodium acetate 20 mm , ph 5 . 0 ) and 50 μl of a tenth dilution was used for injection . total sa was measured by fluorescence ( λ ex 313 nm , λ em 405 nm ) with a nova - pak 4 - mm c - 18 column ( 150 × 3 . 9 mm ; waters ) as part of the waters system ( 1525 binary hplc pump , 2475 multi λ fluorescence detector , 2996 photodiode array detector , 717 autosampler ; waters ). data ( retention time and area ) were analyzed using empower pro software ( waters ). radioactivity was determined by liquid scintillation counting of an aliquot sample . recoveries of the internal standard [ 14 c ] sa were between 20 and 100 % and for each sample , this yield was considered in sa quantity calculation . sa quantity was calculated as followed : ( quantity of sa ( ng )/ fresh weight ( g ))= dilution factor x ([{( area / sa quantity standard curve slope )×( resuspending volume / injection volume )}/ yield ]/ fresh weight ( g )). for ethylene measurements , plants were grown under sterile conditions and leaves were harvested and weighted after two weeks . ethylene was extracted and measured as in [ 35 ]. the mapdisto free software ( http :// mapdisto . free . fr /) was used for qtl and eqtl analysis . two mapping populations were used : a population of 60 rils between moroberekan and co39 [ 38 ] and another between azucena and ir64 with 84 rils [ 39 ]. the gene expression and disease values were log 2 transformed . the distributions of the resulting values followed a normal distribution ( tested with shapiro . test function in r stats package ; data not shown ) and were used for qtl analysis . the goal of this study was to try to establish possible links between the constitutive expression of defense - related genes and partial resistance . our approach was first to evaluate rice diversity ( indica and japonica sub - groups ) for partial resistance . the analysis of partial resistance requires the removal of necrotic , hr - like , lesions that could result from defeated r genes triggering attenuated gene - for - gene resistance [ 32 ]. to meet this criterion , we selected rice / m . oryzae interactions that were as close as possible to compatibility . based on preliminary results ( j l nottéghem , personal communication ), we selected 23 rice cultivars . we also included five rice accessions that are commonly used in the research community and for which genomic and / or genetic information exists ( ir64 , nipponbare , azucena , maratelli and sariceltik ). these cultivars represent 57 % of overall rice allelic diversity , 51 % for japonica sub - group and 55 % for the indica sub - group as estimated by allelic diversity of microsatellites markers ( garris et al , 2005 ). these 28 rice accessions were inoculated with four multivirulent isolates with broad - spectrum virulence ( see methods and fig1 ), and partial resistance was estimated . an index was created for partial resistance that measures fungal growth in planta as quantified by q - pcr ( see methods and fig1 ). in calculating the partial resistance index , we were careful to remove as much as possible background gene - for - gene interactions . these were manifested by extremely low quantities of fungal growth and / or hr - like lesions . out of 25 rice accessions tested , 23 were finally selected as representing most of partial resistance quantitative diversity . the genotypes showing high resistance against all m . oryzae strains ( ct13432 and npe826 ; fig1 and additional file 1 ) were removed from the analysis as they likely reflect complete resistance driven by major r - genes . thus , a total of 23 rice accessions were characterized for partial resistance to rice blast disease and bacterial blight ( fig1 ). from this analysis , it appears that rice accessions from the tropical japonica sub - group were over - represented among accessions with elevated levels of partial resistance to rice blast ( fig1 and 15 ). partial resistance index ranged from 30 ( tropical japonica moroberekan ) to 1 . 3 ( indica padi boenor ). thus partial resistance to blast fungus is highly variable across rice diversity and can vary up to 23 - fold . there was no obvious correlation between partial resistance to blast and bacterial blight . c . constitutive expression of defense as a better indicator of partial resistance than inducible expression we first addressed the question of the relative roles of inducible and constitutive expression of selected defense - related genes in partial resistance . for this purpose , we designed an experiment with a limited number of marker genes ( 11 ) and six representative rice accessions . this experiment was repeated three times independently to monitor gene expression before infection and 1 , 2 , 3 and 4 days post - inoculation ( dpi ). this experiment indicated that most of the defense - related genes selected were induced after infection ( fig2 and 16 ). in order to compare partial resistance to gene expression , we built an expression index that takes into account the expression values of all genes ( see methods and fig1 ). we could then compare the partial resistance index to the expression indexes at the different times before and after infection ( fig3 ). regression analysis ( fig1 ) suggests that there is a good correlation between both indexes before infection ( r 2 = 0 . 8 , p & lt ; 0 . 0027 ) but no statistically significant correlation of these indexes after infection . thus the data on expression of this selected marker genes evaluated on this small set of rice cultivars suggests that expression before infection , more than after infection , correlates with partial resistance . d . the level of constitutive expression of defense - related genes is highly polymorphic across rice diversity we wanted to further extend this analysis to a larger set of rice genes and accessions . we measured constitutive gene expression of 21 genes representative of the rice defense arsenal ( fig1 ) in the 23 rice accessions for which we measured partial resistance . constitutive expression was measured in seven independent experiments at the time when inoculation is usually performed and when partial resistance has started to develop ( 3 weeks after sowing ). when treated individually , the constitutive expression of each gene revealed several points . first , we observed an important variability of the expression levels across cultivars . for example , the expression level of the classical defense gene pbz1 vary up to 35 - fold , with a value of 0 . 02 in one of the most susceptible cultivar , sariceltik , and a value of 0 . 7 in the most resistant cultivar , moroberekan . second , the pattern of constitutive expression was sometimes different between the indica and japonica rice sub - groups ( e . g . the burp gene ). we used hierarchical clustering to identify groups of genes that were co - regulated across rice diversity ( fig4 ). several groups of genes that are co - regulated were found , as supported by bootstrap analysis . the first group ( regulon i ) contains both pr genes and regulatory genes ( pbz1 , pr5 and spl7 ). the second group ( regulon ii ) mostly contains pr genes ( burp , 33 kda , gluc , pox223 and chi ). the third group ( regulon iii ) consists in a last large group of genes that contains both regulatory and pr genes . the last group ( regulon iv ) contains genes involved in recognition ( cebip ) and signal transduction ( mapk6 , hlhdb ). hierarchical clustering of the data also confirmed that the genetic background considerably affects constitutive expression of the selected genes ( fig4 ). for instance , the sub - group of tropical japonica rice appeared clearly different from the other genetic groups of rice . this difference is mostly due to genes from regulons i and ii . thus , rice cultivars from different sub - groups display contrasting capacities to express defense - related genes before infection , suggesting contrasting regulation capacities . e . the level of constitutive expression of defense - related genes strongly correlates with partial resistance across rice diversity it was noteworthy from previous observations that the tropical japonica subgroup is also the genetic sub - group displaying the highest partial resistance index in our analyses ( fig1 ). in order to search for global correlations between constitutive expression of tested genes and the measured partial resistance index , the expression data of the 21 selected defense - related genes in the 23 rice genotypes was analyzed using the expression index already used ( see methods and fig1 ). we found a strong correlation between constitutive expression of defense - related genes and partial resistance ( r 2 = 0 . 83 , p & lt ; 1 . 756e - 6 ; fig5 ). thus , the previous observation on a small subset of rice diversity ( fig3 and 19 ) holds true when tested on a sample of cultivars representing a large subset of rice diversity . similar results were also found when separately testing indica and japonica sub - groups . using principal component analysis and anova ( fig2 ), we could identify the genes that , in our selection , were the most significantly reflecting the correlation between constitutive expression of defense and partial resistance . the pbz1 gene from regulon i was found to be the best marker of constitutive expression of defense - related genes across indica and japonica rice sub - groups . thus , for the pbz1 gene , the observed correlation between constitutive expression and partial resistance holds true for almost all the 23 rice genotypes tested , despite the diversity explored . another gene from regulon i , the spl7 gene , appeared to be a good marker for indica genotypes . the burp and gluc genes from regulon ii were good markers for the japonica sub - group . overall , this analysis across rice diversity suggests that constitutive expression of defense - related genes and partial resistance are highly correlated . partial resistance is well known to increase along plant development [ 19 ]. in particular , in rice there is a strong difference between resistance to blast in a 2 - weeks old plant ( juvenile — susceptible ) and resistance in a 3 - week old plant ( young adult — resistant ). it is also quite common that the last emerged leaf ( leaf n ) is often more susceptible than the leaf that emerged one week before ( leaf n − 1 ). we thus tested whether constitutive expression of defense - related genes was following the same developmental patterns . we chose the tropical japonica cultivars moroberekan and azucena , as they are good representative of constitutive expression of defense - related genes ( fig4 ). constitutive expression of defense - related genes was measured in plants aged from 2 to 8 weeks , on two different leaves ( last and before the last leaf emerged ). as shown in fig6 , the expression of these defense - related genes followed the same developmental pattern than partial resistance with a strong increase in expression between two and three weeks after sowing . this was true for the eight marker genes tested ( data not shown ). this increase of expression was maintained for half of the genes tested . we have yet no explanation for the decrease of expression of some genes like rbbi2 later in development . we also observed that constitutive expression of defense - related genes was overall higher in leaf n − 1 than in leaf n , a pattern very similar to age - related partial resistance . thus , this striking parallel between partial resistance and expression of defense - related genes during plant development further supports our hypothesis that partial resistance can be explained by constitutive expression of defense - related genes . g . constitutive level of sa and ethylene do not explain partial resistance there is a previous report that salicylic acid ( sa ) a signaling molecule involved in disease resistance could play a role in partial resistance of rice to m . oryzae [ 33 ]. jasmonic acid ( ja ) [ 34 ] and ethylene [ 35 ] are also identified as important signaling molecules in plant disease resistance . we asked whether these signaling molecules could relate to constitutive expression of defense - related genes . we evaluated the sa and ethylene pathways by direct quantification of sa and ethylene . we monitored the implication of the ja pathway by using the marker gene rci1 [ 36 ]. the constitutive expression of rci1 did not correlate with partial resistance to m . oryzae ( fig2 ). thus the ja constitutive levels , as monitored by the rci1 gene , do not seem to contribute to partial resistance . sa and ethylene were directly extracted and quantified . the amount of these two molecules was very different across rice diversity ( fig7 ). in each rice sub - group , the constitutive quantities of sa or ethylene were similar in rice accessions showing elevated and weak partial resistance ( fig7 a and 7c ). we could not detect any correlation between the level of partial resistance and the levels of sa or ethylene ( data not shown ). however , we observed that constitutive amounts of sa are 2 - fold higher in indica cultivars than in japonica cultivars and this difference is statistically significant ( fig7 b ). conversely , the constitutive levels of ethylene were higher in japonica than in indica cultivars ( fig7 d ). thus , sa and ethylene constitutive levels negatively correlate ( r 2 =− 0 . 81 , p & lt ; 5 . 2 × 10 − 6 ). although we detected a high level of polymorphism for sa and ethylene in rice , we could not find any correlation between these molecules and partial resistance , nor with constitutive expression of defense - related genes . h . co - localization of qtls controlling constitutive expression of defense - related genes and qtls for partial resistance a prediction of our hypothesis is that we should be able to find areas of the rice genome that control both constitutive expression of defense - related genes and partial resistance . in order to identify such regions , we initiated a qtl analysis on gene expression . expression data has been recently used as quantitative traits for qtl analysis [ 37 ]. the resulting qtls are called eqtls , for expression qtls . two types of eqtls are expected : cis - eqtls that are located at the same locus that the gene monitored for expression ( structural gene ) and trans - eqtls that are located at another locus . we used two japonica x indica mapping populations : the moroberekan x co39 population with 60 recombinant inbred lines ( rils ) [ 38 ] and the azucena x ir64 population with 84 rils [ 39 ]. among the genes tested in this study ( fig1 ), we looked for genes that would show the strongest constitutive expression polymorphism between the parents of the available ril populations ( data not shown ). the burp and chi genes showed the strongest polymorphism and were chosen for eqtl analysis . for each mapping population , two to three independent experiments were done in which constitutive expression of these genes was monitored as well as disease symptoms and used as quantitative traits . the fig8 shows the eqtl and qtl detected with lod & gt ; 3 ( fig2 ) in at least two independent experiments ( false discovery rate of 0 . 001 ). three eqtls ( chromosome 1 , 7 and 11 ) for the burp gene and three eqtls for the chi gene ( two on chromosome 7 and one on chromosome 11 ) were found . most of them were trans eqtls . one cis - eqtl was detected for the chi gene . quite remarkably , two eqtls were common to the chi and the burp genes , suggesting that the constitutive expression of these genes could be controlled by the same locus . the eqtls for burp and chi found on chromosomes 1 and 7 respectively were observed in both mapping populations , further supporting the existence of eqtls in these regions . in all cases the favorable allele increasing constitutive expression was from the tropical japonica parental line ( moroberekan and azucena ). for the burp gene , this is consistent with the observed positive correlation between constitutive expression of this gene and partial resistance in japonica but not indica sub - group ( fig2 ). twelve qtls for blast partial resistance were found ( fig2 ). it was striking that two of these qtls , one on chromosome 7 ( rg4 marker ) and one on chromosome 11 ( rg103a marker ) are co - localizating with eqtl . this is the first genetic evidence that the control of constitutive expression of a defense - related genes could account for partial resistance . global analysis of the variation of gene expression was performed during septoria tritici infection . in the following experiment , p33 kd was identified as a candidate gene for plant response to pathogens infection . two genotypes , cadenza and veranopolis , harboring different stb resistance genes are used . for both genotypes , control and septoria tritici specific type infected plantlets are analysed . rnas from plantlets leaf are extracted and different biological replicates are analysed with genechip wheat genome array ( affimetrix ). p33 kd expression profile is obtained with 3 affymetrix probes : ta . 25531 . 1 . a1_at , ta . 25531 . 2 . a1_at et ta . 25531 . 2 . a1_x_at . the transcriptomic data are presented fig9 and 10 . they show that p33 kd is over - expressed from two days after infection ( dai ) compared to the control . sapi - digested dna fragments encoding the rice ubiquitin 3 promoter , the synthetic gene synos33 kd tamod encoding p33 kd ( seq id no : 3 ) and the terminator atsac66 were ligated into the sap 1 site of the binary plasmid pbios2028 forming the new plasmid pbios2196 ( fig1 ). pbios2028 is derived from plasmid pscv1 , and comprises the gene encoding for nptii under the control of subterranean clover virus scv4 promoter . wheat transformation was performed on nb1 spring wheat variety using an agrobacterium tumefaciens strain containing the pbios2196 plasmid . regenerated transgenic plants have been selected that contain a single insertion locus and an untruncated t - dna from pbios2196 . wheat transformants are grown and analyzed in greenhouse for pathogens resistance . the method is essentially similar to the one described in international application wo 00 / 63398 . wheat tillers , approximately 14 days post - anthesis ( embryos approximately 1 mm in length ), are harvested from glasshouse grown plants to include 50 cm tiller stem ( 22 / 15 ° c . day / night temperature , with supplemented light to give a 16 hour day ). all leaves are then removed except the flag leaf and the flag leaf is cleaned to remove contaminating fungal spores . the glumes of each spikelet and the lemma from the first two florets are then carefully removed to expose the immature seeds . only these two seeds in each spikelet are generally uncovered . this procedure is carried out along the entire length of the inflorescence . the ears are then sprayed with 70 % ims as a brief surface sterilization . agrobacterium tumefaciens strains containing the vector for transformation are grown on solidified yep media with 20 mg / l kanamycin sulphate at 27 ° c . for 2 days . bacteria are then collected and re - suspended in tsim1 ( ms media with 100 mg / l myo - inositol , 10 g / l glucose , 50 mg / l mes buffer ph5 . 5 ) containing 400 μm acetosyringone to an optical density of 2 . 4 at 650 nm . agrobacterium suspension ( 1 μl ) is inoculated into the immature seed approximately at the position of the scutellum : endosperm interface , using a 100 hamilton , so that all exposed seed are inoculated . tillers are then placed in water , covered with a translucent plastic bag to prevent seed dehydration , and placed in a lit incubator for 3 days at 23 ° c ., 16 hr day , 45 μem - 2s − 1 par . after 3 days of co - cultivation , inoculated immature seeds are removed and surface sterilized ( 30 seconds in 70 % ethanol , then 20 minutes in 20 % domestos , followed by thorough washing in sterile distilled water ). immature embryos are aseptically isolated and placed on w4 medium ( ms with 20 g / l sucrose , 2 mg / l 2 , 4 - d , 500 mg / l glutamine , 100 mg / l casein hydrolysate , 150 mg / l timentin , ph5 . 8 , solidified with 6 g / l agarose ) and with the scutellum uppermost . cultures are placed at 25 ° c . in the light ( 16 hour day ). after 12 days cultivation on w4 , embryogenic calli are transferred to w425g media ( w4 with 25 mg / l geneticin ( g418 )). calli are maintained on this media for 2 weeks and then each callus is divided into 2 mm pieces and re - plated onto w425g . after a further 2 week culture , all tissues are assessed for development of embryogenic callus : any callus showing signs of continued development after 4 weeks on selection is transferred to regeneration media mrm 2k 25g ( ms with 20 g / l sucrose , 2 mg / l kinetin , 25 mg / l geneticin ( g418 ), ph5 . 8 , solidified with 6 g / l agarose ). shoots are regenerated within 4 weeks on this media and then transferred to ms20 ( ms with 20 g / l sucrose , ph5 . 8 , solidified with 7 g / l agar ) for shoot elongation and rooting . the presence of the t - dna , and the number of copies are quantified by quantitative pcr ( qpcr ). recombinant wheat plants containing a vector expressing p33 kd are grown and analyzed for pathogens resistance . recombinant rice plants overexpressing p33 kd were generated following a process similar to that described in example j . seq id no : 1 encoding protein p33 kd was cloned into plasmid pubi to generate pubi :: 33 kda ( see fig1 ). transgenic plants ( nipponbare background ) containing overexpression vector pubi :: 33 kda were compared to transgenic plants containing the empty vector after inoculation with magnaporthe oryzae isolate gy11 which is moderately virulent on nipponbare . the number of plants showing either resistance ( no symptoms ), partial resistance ( few lesions but no sporulation ) and susceptibility ( sporulating lesions ) was counted . the results are shown fig1 . they show that the 33 kda population is significantly different from the empty vector population ( p & lt ; 0 . 03 ). data from 35 plants tested ( over - expressor for 33 kda - blue bars ) and 26 control plants for empty vector ( no over - expression of 33 kda - red bars ) expressed as a pourcent . transgenic plants ( nipponbare background ) containing overexpression vector pubi :: 33 kda were also compared to transgenic plants containing the empty vector after inoculation with magnaporthe oryzae isolate fr13 , which is virulent on nipponbare . the number of plants showing either resistance ( no symptoms ), partial resistance ( few lesions but no sporulation ) and susceptibility ( sporulating lesions ) was counted . the results are shown fig1 . they show that the 33 kda population is significantly different from the empty vector population ( p & lt ; 0 . 07 ). data from 29 plants tested ( over - expressor for 33 kda - blue bars ) and 32 control plants for empty vector ( no over - expression of 33 kda - red bars ) expressed as a pourcent . the expression of defense - related genes and molecules is highly polymorphic in rice we decided to use rice diversity in order to establish a role of constitutive expression of defense - related genes in partial resistance . we analyzed 23 rice cultivars that were randomly selected in the two major groups of indica and japonica . these cultivars represent up to 57 % of rice diversity and thus can be considered as a representative sample . in order to evaluate partial resistance for magnaporthe oryzae , we generated an index that mostly takes into account fungal growth . complete resistance driven by specific gene - for - gene interactions was removed . overall , our quantitative index correlates with other measurements of partial resistance like lesion number ( xg and jbm , data not shown ). partial resistance to blast fungus did not correlate with quantitative resistance to rice blight ( fig1 ). this may be due to different lifestyles of the fungal pathogen ( hemibiotrophic , growing in the mesophyl ) and bacterial pathogen ( biotrophic , growing in the xylem ) tested . this may also be due to the fact that partial resistance to blast was evaluated on 3 - weeks old plants whereas resistance to bacterial blight was evaluated on 8 - weeks old plants . the genes that were used to measure constitutive expression are representative of the disease resistance pathway . most of the regulators have been demonstrated to be positive regulators of resistance by mutant analysis ( see references in fig1 ). many of the defense genes studied have also been shown to increase resistance in plants that are over - expressing them [ 25 ]. finally , the osks4 gene was selected as a representative gene for the momilactone biosynthetic pathway , one of the major rice phytoalexin [ 40 ]. it was clear from our analysis that constitutive expression of the defense - related genes selected was highly polymorphic across rice diversity ( fig2 ). this analysis revealed that the tropical japonica sub - group displays a unique capacity to express defense - related genes before infection ( fig4 ). this observation likely reflects that these genotypes possess polymorphic and / or unique regulators of disease resistance . the constitutive amounts of signaling molecules like salicylic acid and ethylene is also extremely polymorphic ( fig7 ), especially between indica and japonica cultivars . comparing this polymorphism to expression polymorphism of other plant metabolic pathways would tell us whether these elp ( expression level polymorphism ) are comparable or not . an analysis in arabidopsis suggests that defense - related genes display elevated elps as compared to genes belonging to other pathways [ 29 ]. it is also noteworthy that sequence polymorphism of defense - related genes is low in arabidopsis [ 42 ]. it is thus likely that disease resistance polymorphism results more from expression than from polymorphism at the protein level , with the exception of gene - for - gene polymorphism . preformed defense , but not induced defense , is a hallmark of partial resistance to the rice blast fungus our hypothesis was that the constitutive expression of defense - related genes was contributing to partial resistance . this was initially based on observation on a few rice cultivars and genes ( ev and jbm , data not shown ). this was also motivated by several piece of literature reporting that physical barriers [ 20 , 22 ] and preformed antimicrobial molecules [ 23 ] play an important role in disease resistance . we thus addressed the question of a putative contribution of defense - related genes in general in partial resistance . we built an index of gene expression where all genes participate to the same extent to the final value ( see methods and fig1 ). when compared to the index of partial resistance , we found a very strong correlation between constitutive expression of defense - related genes and partial resistance ( fig5 ). this correlation was extremely robust as we observed it in seven independent experiments in the past two years . we conclude that this constitutive expression of defense - related genes is a preformed defense system that contributes to partial resistance . the group of tropical japonica cultivars showed an atypical pattern of constitutive expression of defense - related genes ( fig4 ). these cultivars also displayed a high index of partial resistance and of constitutive expression of defense . this observation suggests that this rice sub - group harbors a particular preformed defense system . some genes like the regulatory gene spl7 and the pr gene pbz1 were good markers of preformed defense ( fig2 ). the only prr gene tested here , the cebip gene , was not a good marker of preformed defense . cloned r genes [ 18 ] and more recently identified receptor - like genes ( waki ) [ 43 ] need to be tested to determine if constitutive expression of defense - related genes involves all steps of the basal and gene - for - gene resistance pathways . finding more genes with an expression pattern correlated to partial resistance will help us to build gene sets to further studying preformed defense . there was no obvious correlation between partial resistance and expression after infection of the defense - related genes tested ( fig3 and 19 ). since the infection process of rice by m . oryzae occurs very early after inoculation , it is possible that we underscored early time points ( less than 24 h after infection ). it remains possible that gene expression in the very early steps of infection also correlate with partial resistance . it is also extremely difficult to estimate the part of partial resistance that can be attributed to preformed and induced defense . identifying genes that control preformed but not induced defense could help defining the respective contribution of each system . without rejecting the probable contribution of induced defense in partial resistance , our results strongly suggest that constitutive expression of defense - related genes highly contributes to partial resistance . one of the best evidence for a contribution of preformed defense to partial resistance comes from the observation that these two phenomena are coordinated during development . partial resistance is well known in rice to be developmentally regulated [ 19 ]. when we measured constitutive expression of defense - related genes , we observed that this expression was following the increase of partial resistance during plant growth . the constitutive expression of all genes tested dramatically increase between juvenile ( 2 - week old plant ) and young adult plants ( 3 - week old plants ) ( fig6 ). such a massive effect suggests that there is a major control of development on the expression of preformed defense . recently , zhao et al [ 45 ] also observed that the constitutive expression of the r genes xa3 / xa26 and xa21 were developmentally controlled . this could easily explain in this case why gene - for - gene resistance driven by these genes was effective in adult plants but not in juvenile plants . finding common regulatory points between development and defense may help us understanding how partial resistance is developmentally controlled . in order to get further insights on the way preformed defense is deployed by rice , we tested the implication of three signaling pathways controlled by salicylic acid , jasmonic acid and ethylene in constitutive expression of defense - related genes . we did not find evidence that these pathways were involved . this was unexpected for the sa pathway since previous report [ 33 ] suggested that constitutive sa levels were correlated to m . oryzae resistance . when we closely examined this report , we found out that the disease index used by silverman and colleagues was not defined and that disease resistance in their case most likely correlated with indica / japonica differences . this could simply reflect the fact that some m . oryzae isolates are better adapted on one rice sub - group than on another . we circumvented this difficulty by trying to incorporate in our disease index only partial resistance as monitored by using multivirulent isolates of m . oryzae . we conclude that neither constitutive expression of sa , ja nor ethylene pathway correlates with elp of defense - related genes . alternatively , preformed defense could result from the leakage of the disease resistance pathways . for example , assuming that some signaling is constantly triggered by the environment , rice cultivars having efficient but leaky regulatory pathways would also display elevated levels of defense - related genes in the absence of infection . this mechanism would require positive regulators of disease resistance to be very active and negative regulators to be quite inactive . this mechanism would be similar to the mechanism by which the barley m / o gene confers resistance to powdery mildew . the mlo gene is a negative regulator of disease resistance and recessive alleles ( m / o ) of this gene confer broad - spectrum resistance [ 46 ]. some m / o alleles are weak negative regulators such that the plant constitutively expresses parts of the disease resistance pathway , leading to spontaneous cell - death that resembles hr [ 47 ]. forward genetics is one way to identify the genes that regulate preformed defense . using qtl mapping , we show that preformed defense is amenable for genetics . several regions of the rice genome controlling preformed expression of the chi and burp genes were identified ( fig8 ). the architecture of this control is probably complex since our analysis of only two genes revealed six eqtls . however , the observation that the burp and chi belong to the same regulon ( fig4 ) is consistent with the observation that two eqtls are common to these genes ( fig8 ). these regions of chromosome 7 and 11 may contain regulators of preformed defense that are specific to the tropical japonica sub - groups . fine mapping of these regions will help us identify the genes that control preformed defense and partial resistance . more importantly , we show the first genetic evidence that two eqtls controlling constitutive expression of the chi and burp genes co - localize with two qtls for partial resistance . given the number of genetic markers used for mapping ( 133 ), the number of qtl ( 11 ) and eqtl ( 6 ) found , the probability to find such co - localizations was very low ( p = 0 . 011 ). a more detailed analysis will be necessary to establish a functional relationship between these two phenomena . interestingly , one of the eqtl controlling chi constitutive expression co - localizes with the chi structural gene on chromosome 7 . thus this eqtl could be a cis - eqtl . we did not find a potential cis - eqtl for the burp gene , suggesting that constitutive expression for this gene is mostly controlled in trans . given the yet imprecise position of the eqtls , the eqtl controlling the chi around the rg4 marker could also be a trans eqtl . in such a case , this region around rg4 marker on chromosome 7 could be a common regulator of constitutive expression for burp and chi . our qtl analysis already pinpoints some regulatory candidates that co - localize ( 4 mb range ) with several eqtls . the eqtl on chromosome 1 co - localizes with oswrky13 [ 49 ]. this transcription factor has been shown to be involved in blast disease resistance as plants over - expressing oswrky13 show enhanced resistance to this pathogen . plants over - expressing oswrky13 also displayed constitutive , elevated , levels of expression of defense - related genes but pbz1 was down - regulated . thus this gene is unlikely a good candidate for regulating preformed defense . the eqtl on chromosome 7 ( close to the rg4 marker ) co - localizes with the osdr8 [ 41 ] and the cigr1 [ 48 ] genes . preliminary analysis of the cigr1 mutant suggests that this gene is not responsible for the eqtl ( blein m , xg and jbm , data not shown ). the osdr8 gene is involved in the vitamin b1 biosynthesis pathway and in thiamine accumulation [ 41 ] is also found within 4 mb of the rg4 marker on chromosome 7 . interestingly , plants silenced for osdr8 show increased susceptibility to m . oryzae and reduced accumulation , before infection ( as well as after infection ) of several pr genes , including pox223 but not pbz1 . this is only partly consistent with a possible role of osdr8 in preformed of defense . consistent with the implication of the thiamine pathway in preformed defense , thiamine is known to be an inducer of defenses in plants , including rice [ 44 ]. finally , the eqtl close to marker rg351 on chromosome 7 co - localizes with the rtga2 . 1 gene [ 55 ]. although silencing of the rtga2 . 1 gene increased the constitutive expression of defense - related genes , it is yet unknown whether this mutation affects resistance to m . oryzae . such attempt to co - localize known regulatory genes with eqtl is overall risky and fine mapping will be required to identify the genes explaining these eqtls . preformed defense systems as a way to respond to environmental stresses in plants plants have evolved sophisticated inducible systems to respond to pathogen challenge [ 1 ]. expression level polymorphism ( elp ) has been shown to be important for the gene - for - gene resistance pathway [ e . g . 30 , 31 ] but there was up to now no indication that elp could play a role in partial resistance . by looking at elp in partial resistance of rice to m . oryzae , we provide several lines of evidence that constitutive expression of defense - related genes correlates with partial resistance in naturally occurring diversity . this is the first evidence of the role of constitutive expression of defense - related genes in disease resistance . plants have deployed such a proactive strategy to face abiotic stresses [ 50 , 51 ]. for example , a large portion of the genes that are normally induced by zinc stress in arabidopsis thaliana are constitutively highly expressed in a . halleri , a species of the arabidopsis genus showing enhanced tolerance to zinc . thus , constitutive expression of zinc - responsive genes has been proposed as a mechanism by which a . halleri naturally increases its tolerance to zinc [ 50 ]. using a similar approach , taji et al [ 51 ] showed that a large number of abiotic or biotic stress - inducible arabidopsis thaliana genes were expressed under normal growth conditions in salt cress ( thellungiella halophila ), a naturally salt tolerant plant specie . thus plants seem to have evolved proactive , non - inducible systems to face abiotic stresses . therefore , it appears that constitutive expression of the adapted repertoire of genes is a general strategy used by plants to face environmental pressure . this is consistent with our current knowledge on trait evolution which poses that regulatory polymorphism might better account for phenotypical variability than structural polymorphism [ 52 ]. past research has largely focused on inducible mechanisms to explain disease resistance . we provide three lines of evidence that constitutive expression of defense - 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