WO2017063060A2

WO2017063060A2 - Method for production of transgenic plants and bacteria-resistant plants having the "quorum sensing" system

Info

Publication number: WO2017063060A2
Application number: PCT/BR2016/000068
Authority: WO
Inventors: Carlos Eduardo FERMINO
Original assignee: Instituto Agronõmico Do Paraná - Iapar
Priority date: 2015-10-14
Filing date: 2016-07-14
Publication date: 2017-04-20
Also published as: BR102015026078A2; WO2017063060A3

Abstract

The present invention is related to a method for production of transgenic plants resistant to bacteria having the Quorum sensing system and transgenic citrus plants resistant to the Huanglongbing or HLB disease. The aiiA gene, which encodes the AHL-lactonase enzyme and which is shown is SEQ ID n° 1, was used for transformation against HLB. This enzyme inactivates the acyl homoserine lactones (AHLs) produced by several phytopathogenic bacterial species, preventing this communication component (Quorum-sensing) from functioning in transgenic plants, and potentially preventing the development of the disease in the inoculated plants, We claim to have transgenic citrus trees containing the aiiA gene that are tolerante to HLB, and to other bacterial plant disease having the "quorum sensing" system.

Description

METHOD FOR PRODUCTION OF TRANSGENIC PLANTS AND BACTERIA-RESISTANT PLANTS HAVING THE "QUORUM^' SENSING"

SYSTEM

Field of the invention

The present invention is related to the method for providing resistance to the ability of communication between bacteria having the Quorum sensing system, such as Candidatus Liberibacter sp. species and others. The invention is in the field of agriculture and genetic engineering, more specifically, of new plants and methods for obtaining them, as it refers to a method for the production of transgenic plants resistant to bacteria having the Quorum sensing system and transgenic plants resistant to Huanglongbing disease or HLB and others having the Quorum sensing system.

Background of the invention

Huanglongbing, ox HLB, is the official name of the citrus plant disease which has great importance woridwide nowadays, but was restricted to the Asian and African continents until the rnid-2000's. It was detected in the state of Sao Paulo and Parana, Brazil, respectively in 2004 and 2005, and subsequently in the American state of Florida in 2005. HLB has caused huge losses in these areas as if significantly reduces yield and quality of the remaining fruits. It is usually deemed as the main problem for fruit production, as it affects all commercial citrus plants, such as limes, oranges, lemons, tangerines and related cultivars, and there is no natural genetic resistance against this disease within these species.

HLB is associated with the presence of the not yet cultured bacteria Candidatus Liberibacter spp. and 2 species (C.L, asiaticus and C.L.

americanus) have been detected in Brazil so far. Only the Asian variant is present in Florida, in Africa, there is the African strain. All of them are aggressive and have large potential to cause damage to host citrus trees. These bacteria live inside the phloem vessels of citrus plants, and these vessels are responsible for redistributing sugars, hormones and many other compounds which are essentia? to the host piants. Therefore, these bacteria are lodged in a nutrient-rich tissue, and perhaps because of this, they can cause symptoms even at low concentrations under experimental conditions.

The official name HLB (Huang!ongbing) was proposed in a convention of virologists as reference to the problem described in China since the end of the 19th century, where producers observed yellow branches in affected trees. The name "Greening" is used in South Africa and in other countries because of the greenish and irregular coloring of fruits in affected trees.

Liberibaeters can be carried to new piants by winged insect vectors named Diaphorina citri Kuwayama in America and Asia continents and Trioza eryireae (Del Guercio) in Africa. They belong to the group of psyliids, which feed themselves on the phloem of citrus plants. These insects without the presence of bacteria cause no economic damage to citrus piants, but when earring them, the damage caused by the disease can be severe, making economical production of fruits very difficult in many cases. The bacteria not only use the insect as a mean of transportation, but they are also able to reproduce themselves, increasing their populations significantly within the host insect, !n simple terms, the bacteria use the insect as transportation and temporary residence. Such pathosystem in citrus plants can be considered similar to Dengue feve in humans. Apparently, the bacteria do not harm the insects, which in addition of being efficient vectors, they can fly and can be carried by air currents at large distances. A harmful consequence of all these features is that the disease can be spread over long distances, not yet precisely determined, but certainly transcending the boundaries of farms, regions, and countries with high efficiency. Under experimental conditions, the Asian variant has been detected in test plants only 30 days after inoculation at a distance of 2 km, while the visual symptoms only appeared after 3 or 4 months (in most cases, it takes between five and six months), !n these experiments, inoculated plants presenting no symptoms yet were also capable to transmit the disease via side grafting of budwoods from asymptomatic but infected branches.

Under field conditions, farmers will always pursue to mitigate the progress of the disease since infected plants having no symptoms go unnoticed, but are already source of inoculum. Therefore, it is essential that controifing actions must be at least quick and efficient. Currently, the control of this complex disease is based on planting healthy trees, and on the reduction of the inoculum by the elimination of symptomatic plants in the field (with huge losses for farmers) and by the difficult attempt to controi the insect vector through spraying pesticides, which in many cases are carried out on a weekly basis (with serious environmental and food safety impacts). Despite those efforts, the progress of the disease was notorious both in Brazil and in U.S.A. in recent years, showing that the current methods were not effective against the disease. Therefore, it becomes imperative to create resistance to this disease using genetic engineering.

There are no reports of commercial varieties of citrus plants being naturally resistant to this disease, making the efforts to perform classical breeding fay crossing plant species very difficult. On the other hand, the use of genetic engineering provides a much broader approach in the creation of strategies against HLB. Liberibacters have a mechanism of communication between individual cells designated Quorum sensing., which is used to determine the population density in the environment. This perception causes bacteria to alter their gene expression levels, activating and/or deactivating groups of genes able to stimulate cells multiplication and virulence, causing damage to the host plant.

The present invention provided transgenic citrus plants capable of producing an enzyme to chemically breakdown the molecule used as the exchanged signal by bacteria in the phloem. Those transgenic host plants acquired an enzymatic defense reaction that mitigates the bacteria's Quorum sensing system, causing them to lose information on population density. This mechanism minimizes the bacteria! multiplication process and reduces the appearance of symptoms and damage in the affected plants. This strategy does not prevent the infection of the host plant by

Lsberibacters, since it would be impossible to be done due to the

undetermined amount of contagious psyilids dispersed throughout productive areas, but reduces the multiplication of bacteria and creates HLB tolerant plants.

Any effective mechanism of genetic resistance to HLB will provide great resource and energy savings to the citrus industry, food safety for the overall population and considerable environmental advantages due to the reduction of pesticide applications on orchards. HLB is a devastating plant disease that has turned the general orchard management very difficult to economically produce citrus fruits in various regions in Florida, Sao Paulo and other regions worldwide.

State of the art

The document "WO2014/174474-A2" ("Plants resistant to

pathogenic microorganisms growing in vascular tissues", by Cazares et ah, 2014) describes transgenic plants resistant to infections caused by microorganisms limited to the phloem, comprising the expression of a fusion or a chimeric protein - a Citrus aurantifolia (CsPPIS) phloem protein, a protein binder and a protein having antimicrobial activity. The present invention is not in conflict with this document, as the proposed transgenic plants only use a protein that prevents bacteria from communicating with each other, creating an indirect effect on them for making multiplication thereof difficult. There is no direct antimicrobial activity, the proposed invention while the said document does have this antimicrobial activity.

The document "US2013/02D5443-Ar ("Pathogen resistant citrus compositions, organisms, systems and methods", by Mirkok &

Gonza!es-Ramos, 2013) reports compositions, organisms, systems, and a method for increasing the inherent ability of plants (e.g.: citrus plants) to respond to the contact with a pathogenic agent. This document is related to defensin protein gene, which is a class of protein with direct action on the bacterium, and therefore, it is not related to the present invention. The document "Response of Citrus Transgenic Plants Expressing STX IA Gene to Candidatus Liberibacter assaticus" (by Marques et at, 2011 ) describes transgenic citrus plants transformed with the STX !A gene and a study of the resistance to HLB disease. The STX gene has a direct antimicrobial activity, acting on the bacteria! ceil membrane. The

mechanism of action is different from that proposed herein.

The document "Desarroilo de alternativas biotecnoJogicas para ia obtencion de plantas de Citrus sinensis (L.) Osbeck resistentes a la

enfermedad de ia Cancrosis de los citricos" (by Furman, 2013) describes transgenic orange plants (Citrus sinensis L Osbeck) resistent to cancrosis by expressing the antimicrobial peptide dermaseptin. This document is not in conflict with the present invention since the antimicrobial protein dermaseptin is an antibiotic drug of direct action and is mentioned to be used against Xanthomonas, which causes another disease designated Citrus canker, while the transgenic plants proposed herein use a single protein that prevents bacteria from communicating with each other, as an indirect effect for the treatment of another disease, HLB.

The document "Breeding citrus for HLB resistance at the USDA/ARS U.S. Horticultural Research Laboratory, Ft. Pierce, Florida" (by Stover et al., n/d)relates transgenic strategies for the development o f HLB resistant citrus plants. Such strategies are related to antimicrobial antibiotic proteins that kills bacteria by iysing the cell membrane, or acting on the respiration process by oxidative damage. This is different from the strategy used in the present invention.

The document "US2015/0067918-A1" ("Citrus plants resistant to citrus huang!ongbing (ex greening) caused by Candidatus. Liberibacter asiaticus (las) and bacterial canker caused by (Xanthomonas axonopodis pv. citri) (xac)", by Kress, 2015) is related to viral vectors based on modifications of the Citrus Tristeza virus (CTV) useful for transfecting citrus trees for beneficial purposes, including control of HLB. The method uses CTV genes assembled with Green Fluorescent Protein gene (GFP), lectin gene from Allium sativum (ASL), agglutinin gene from Pineflia ternata (PTA) and others. There is no relation with the lactonase enzyme (aiiA gene) used in the present work.

Brief description of the invention

The present invention relates to a method for the productio of transgenic plants resistant to bacteria having the Quorum sensing system and transgenic plants resistant to HLB disease and other bacteria having the Quorum sensing system- The aiiA gene, which encodes the

AHL-lactonase enzyme, was used for transformation against HLB. The gene is shown in SEQ ID n° 1. This enzyme inactivates the acyi homoserine lactones (AHLs) produced by phytopathogenic bacteria, preventing this communication component (Quorum-sensing) from functioning in transgenic plants, and potentially preventing the development of the disease in the inoculated plants. The aiiA gene was assembled under control of the CaMV 35S (Cauliflower Mosaic Virus 35S) constitutive promoter, shown in SEQ iD n° 2. Transformations were made in explants of thin cross sections of the epicotyl of piantiets grown in vitro and from tissues of mature branches with co-cultivation with Agrobacterium tumefaciens, strain EHA 105, containing the plasmid with the transgene of interest in accordance with the used construction. Explants were selected in a medium containing antibiotics. The presence of the transgene was confirmed in sweet orange plants (Citrus sinensis L. Osbeck), Swingle citrumelo (Citrus paradisi acf. x Poncirus trifoliata L. Rat) and Citrange Carrizo (Citrus sinensis L. Osb. X Poncirus trifoliata L. Raf.).

Brief description of the figures

Figure 1 shows the vector map with the construction PBi- At representing the following regions: Ca V35S promoter (Cauliflower Mosaic Virus 35S promoter), NPTII (Neomycin Phosphotransferase If) selection gene that confers resistance to kanamycin, the gene of interest aiiA

encoding the AHL lactonase enzyme and the NQS (nopa!ine synthetase) terminator. Figure 2 shows DNA fragments amplified by PGR reaction

(Polymerase Chain Reaction) of transgenic plants (numbers 7916 to 7996) containing the gene aiiA of interest; The first lane on the left represents a DNA molecular marker weight, plant 7914 Is non-transgenic; lane "aiiA" is the positive control of the reaction and the la ne "blank" is the negative control.

Figure 3 shows the side grafting of the infected bud used as HLB inoculum from a. symptomatic Valencia' orange tree found in the field and grafted on the stem of the transgenic plant (Citrange Carrizo), candidate to be resistant to HLB.

Figure 4 shows a plant containing symptomatic leaves of the inoculum buds side grafted on the stem of the transgenic plant whose shoots have exhibited no symptoms of HLB.

Figure 5 displays, the relative concentration of bacteria present in each sample, showing far less relative bacteria on the transgenic than on the non-transgenic plants.

Detailed description of the invention

The present invention provides a method for production of transgenic plants resistant to bacteria having the Quorum sensing system and transgenic plants resistant to HLB disease and others having the Quorum sensing system.

The Quorum sensing system is present in some bacterial species being used by them to assess the relative amount of bacteriaS cells present in the environment. Bacteria having this system have a set of genes that express signals, such as lactone, which are exported out of the cell and are quantified by bacteria within the same environment. It causes the bacteria to be able to notice their relative amount.

Occurrence of diseases i plants caused by bacteria having the Quorum sensing system is related to the titer of these bacterial ceils in the host. Therefore, the mitigation of the signals (lactones) emitted by these bacteria disturbs their ability to reproduce. The invention comprises introducing the aiiA gene into plants_., thai starts to express the iaetonase enzyme, which is responsible for breaking down lactones, drastically reducing the ability of the bacterial cells to cause the HLB disease, and other diseases that also use the Quorum sensing system to communicate v/ith each other.

The method has steps consisting of the steps of:

I. Providing plant cells with a chimeric DNA molecule comprising a first and a second regions where;

1.1 The first region comprises a nucleotide sequence having at least 243 consecutive nucleotides, which have substantial similarity with a sequence having about 243 consecutive nucleotides in the sense region of the gene {aiiA) coding for an enzyme of th Iaetonase peptide biosynthetic pathway, as shown in SEQ ID n°^' 1 ;

1.2 The second region comprises a nucleotide sequence having at least 195 consecutive nucleotides, which have substantial similarity with the complementary sequence of about 195 consecutive nucleotides of the sense region of the Ca VSSS biosynthetic promoter, as shown in SEQ ID n° 2;

1.3 The first and second regions are capable of forming a spacer region between them; and

IS. Identifying those plants where the expression of the enzyme from the Iaetonase peptide biosynthetic pathway is then regulated.

In step I the plant is selected from the groups of the Rutaceae Juss family, Aurantioideae sub-family, Citreae and CSauseneae tribes, and subtribes comprising genera Citrus sp., Poncirus sp., Fortunella sp., Murraya sp., Severinia sp. Microcitrus sp., Atalaniia sp., and hybrids thereof.

In one aspect of th present invention, the proposed method is used to transform plant cells from the genus Citrus sp., Poncirus trifoiiata L Raf. and hybrids thereof with a nucleic acid molecule expressing the !actonase peptide. The expression of the said peptide provides resistance to bacterial pathogen having the Quorum sensing mechanism.

Th DNA molecule comprises gene regions encoding an enzyme of the lactonase biosynthetic pathway, wich is the active ingredient for mitigating the bacterial Quorum sensing system.

Transformation comprises infecting the plant cell with Agrobacterium tumefaciens and/or microprojectile bombardment harboring the said nucleic acid expressing the Lactonase peptide.

The present invention further refers to a chimeric DNA molecule for expressing the target gene in a plant cell comprising:

I. A promoter region capable of being recognized by RNA

polymerases in plant cells, linked to:

II. A DNA region which, when transcribed, produces a mRNA molecule comprising RNA regions specific to the gene containing:

a) A region comprising a nucleotide sequence having at least 243 consecutive nucleotides, which have substantial similarity with a sequence having about 243 consecutive nucleotides of the sense region of the gene coding for an enzyme of the lactonase peptide biosynthetic pathway, as shown in SEG ID n° 1 ;

The used gene that expresses the lactonase enzyme was obtained from soil bacillus, as described in. the paper by Dong et al 2000, and available at the Genebank under accession number AF198488. It was assembled on a chimeric DNA sequence containing a promoter (Ca V35S) and a selection gene (Λ/Ρ71Ι). The construct was introduced into

Agrobacterium tumefaciens and used in the genetic transformation of citrus plants.

Agrobacterium tumefaciens comprises the pBl-AI construct containing the aisA gene which codes for the AHL lactonase enzyme, a nopaline synthetase (NOS) terminator and a kanamycln resistance gene (NPTll, or neomycin phosphotransferase II), under control of the CafvlV35S promoter, as shown in SEQ ID n° 2.

Also, the present invention includes transgenic plants comprising the aHA gene encoding the AHL-lactonase enzyme that breaks down signals produced by pathogenic bacteria, mitigating the Quorum sensing system thereof and causing resistance to HLB and similar diseases. It reduces the ability of bacteria, to reproduce, enabling adequate development of the infected plants.

The said transgenic plants were seiected from groups of Rutaceae Juss family, Au.rantioideae sub-family, Citreae and Clauseneae tribes, and subtribes comprising genera Citrus sp., Poncirus sp., Fortunella sp.,

Murraya sp., Severinia sp. Microcitrus sp., Ata!antia sp., and hybrids thereof.

The transgenic plants are transformed with a nucleic acid molecule expressing the lactonase peptide. The expression of that peptide provides resistance to bacterial pathogens having the Quorum sensing mechanism, thereby inducing a phenotype of tolerance to diseases which pathogens have the Quorum sensing system, such as HLB {Huanglongbing) and other diseases.

The present invention also includes the progeny, the rootstock, the somatic embryo, the radicular, apical, bud and side meristems, the cell, and the seeds of the transgenic plants.

Detailed aspects of the created invention

Epicotyl segments of Citrus sp., Poncirus trifoiiata L. Raf, and their hybrids were genetically transformed with EHA105 Agrobacterium tumefaciens strain containing the pBI-AI construct (Figure 1) and received the gene of interest aHA, shown in SEQ ID n° 1. Figure 2 shows DNA fragments of the transgenic plants with the insert of the transgene amplified by PGR reaction (Poiirnerase Chain Reaction).

These plants were subjected to two phases, the first one comprised in vitro co-cuitivation of Citrus sp. expiants exposed to Agrobacterium tumefaciens. In the second step, the selected and candidate expiants to be transgenic were established in pots in greenhouses for 2 years.

The transgenic materials were multiplied and were then challenged for HLB pressure. Buds of affected plants collected from the field were side grafted on the main branch of transgenic plants candidates for developing resistance (figure 3).

Twenty-four months after inoculation, under controlled greenhouse conditions, the transgenic plants did not show symptoms of the disease, while the inoculum buds already exhibited the typical symptoms of HLB (figure 4).

AH the transgenic plants challenged with HLB were subjected to Real-Time PGR diagnostic tests for quantitative assessment. Table 1 shows the results for Citrange Garrizo.

The experiment consisted of side grafting an infected bud from a branch showing typical symptoms of HLB of a Valencia orang tree from the field to the stem of Citrange Carrizo transgenic plants, in order to transmit the disease to the transformed plants. Therefore, the same tested plant contained both the transgenic and the HLB contaminated material

(inoculum), providing ideal conditions for disease contamination by contact of tissues.

Analysis of the visual symptoms and the relative quantification of the causative agent of HLB was performed from methods that have already been described in the literature, consisting of visual analysis for the patterns of asymmetric chlorosis on leaves and amplification of the bacterial target DNA in the assessed plant tissue. A symmetrical chlorosis on leaves of plants with HLB was used to assess the presence or absence of symptoms^' of the disease on the plant. Amplification of the bacterial target DNA by real-time PGR was used for relative quantification of the causative agent of HLB, i.e., bacterium Candidatus Liberibacter asiatics, in the assessed plant tissue (table 1). While the inoculum bud has developed the typical symptoms of the disease, the bud from the transgenic material did not present any symptoms (figure 4).

Table 1: Quantitative assessment of transgenic plants challenged with HLB

Analysis by real-time PGR provides quantitative information about the amplified target DNA. Values designated "Ct" refer to the number of amplification cycles of the target DNA that enabied the detection of a certain fluorescence level of the DNA probe used to amplify the Canditatus Liberibacter sp DNA. Low Ct values mean that the PGR reaction has started before another sample having higher Ct value, that is, there was a higher amount of target (bacterial DNA) than the other sample. The difference betwee the two Cts is proportional to the difference in concentration between the amplified targets. It is calculated by the exponential function f(x)=2n, wherein variable "n" is the difference of Cts between two samples. Fo example, plant no. 6869 has an average Ct value of sample "1 " of 33.56 and of sample "2" of 23.62. The difference between them is ACt=33.56 - 23.62 = 9,94, that is, sample "2" (inoculum) has f(x)=29,94 = 982-fold more bacteria than sample "1" (transgenic Carrizo estrange).

Plant no. 6621 showed a Ci difference of ACt=29.02^■■■· 23.1 1 = 5.91 , that is, sample "2" (inoculum) has f(x)-25.91- 60~fold more bacteria than sample "1" (transgenic Carrizo Citrange).

ft .can also be noted that the average Ct of the endogenous GAPDH (G!ycera!dehyde 3-phosphate dehydrogenase) control, as expected, did not show a large variation in amplification.

In another experiment, the relative amount of bacteria present in each sample was evaluated (figure 5). In fhatanalysis, the non-transgenic control (plant 6150) was used as a normalizer to serve as a reference for high infection, and, therefore, was deemed to be 100%. The further transgenic plants (7908 to 18,594) containing the ailA gene that codes for the AHL lactonase enzyme had much lower levels of Canditatus

Uberibacter sp.(C.Las.): 13.68% for plant 7908; 5.30% for plant 7937; and lower than 1.6% for the remaining plants. Sample 1G+Las was the positive control of the reaction and H20 was the negative control.

Transgenic carrizo plants 7915, 6572 and 18,595 had very low levels of bacterial concentrations of as low as 0.05% over the non-transgenic normalizer control ( 00%) and did not show any visual symptoms. The general normalizer used was the non-transgenic plant 6150 used for the relative comparison of results. The negative control (Η2Ό) obtained an undetermined Ct value, being considered as zero or negative, and 1C+L,as. was the positive control. Inoculums exhibiting visual symptoms also presented high bacterial concentration levels, as determined by real-time PGR, while transgenic plants containing the Lactonase enzyme expressed by the gene of interest aiiA, that breaks down the signals produced by Liberibacters to mitigate the Quorum sensing system thereof, reduced the ability of multiplication of HLB bacteria, thereby providing a satisfactory development of the infected plants.

Claims

1. A method for obtaining transgenic plants having resistance to bacteria with the Quorum sensing system characterized by comprising the steps of:

!. Providing plant ceils with a chimeric DNA molecule comprising a first and a second regions where;

1.1 The first region comprises a nucleotide sequence having at least 243 consecutive nucleotides, which have substantial similarity with a sequence havsng about 243 consecutive nucleotides in the sense region of the gene coding for an enzyme of the lactonase peptide biosynthetic pathway, as shown in SEQ ID n° 1 ;

1.2 The second region comprises a nucleotide sequence having at least 195 consecutive nucleotides, which have substantial similarity with the complementary sequence of about 195 consecutive nucleotides of the sense region of the CaMV35S biosynthetic promoter, as shown in SEQ !D n° 2:

1.3 The first and second regions ar capable of forming a spacer region between them; and

li. Identifying those plants where the expression of the enzyme from the lactonase peptide biosynthetic pathway is then regulated.

2. The method of claim 1 , characterized in that the plant is selected from the groups of Rutaceae Juss family, Aurantioideae sub-family, Citreae and Clauseneae tribes, and subtribes comprising genera Citrus sp., Poncirus sp., Fortunella sp., Murraya sp., Severinia sp. Microcitrus sp., Atalantia sp., and hybrids thereof,

3. The method of claim 1 , characterized by transforming a cell of a plant of the Rutaceae Juss family, Aurantioideae sub-family, Citreae and Clauseneae tribes, and subtribes comprising genera Citrus sp., Poncirus sp., Fortunella sp., Murraya sp., Severinia sp. Microcitrus sp., Atalantia sp., and hybrids thereof with a nucleic acid molecule expressing the lactonase peptide, with the expression of the said peptide provides resistance to bacteria! pathogen having the Quorum sensing mechanism.

4. The method of c!aim , characterized by the DMA molecule comprising multiple regions of the gene encoding an enzyme from the lactonase peptide biosynthefic pathway.

5. The method of claim 4, characterized by the enzyme from the lactonase peptide biosynihetic pathway, the active ingredient of the Quorum sensing system.

6. The method of claim 1 , characterized by the transformation which comprises infecting with Agrobacterium tumefaciens and/or microprojectile bombarding said plant cell comprising said nucleic acid expressing said Lactonase peptide.

7. A chimeric DMA molecule for expressing the target gene in a plant cell, characterized by comprising:

!. A promoter or promoter region capable of being recognized by NA polymerases in plant cells, linked to:

II. A DNA region which, when transcribed, produces a mRNA molecule comprising RNA regions specific to the target gene containing: a) A first region comprising a nucleotide sequence having at least 243 consecutive nucleotides, which have substantial similarity with a sequence having about 243 consecutive nucleotides of the sense region of the gene coding for an enzyme of the lactonase biosynthetic pathway, as shown in SEQ ID n.° 1 ;

b) A second non-transcribed DNA region comprising a

complementary sequence having about 95 consecutive nucleotides of the sense region of the biosynthetic CaMV35S promoter, as shown in SEQ ID n° 2;

c) A first and second regions capable of forming a spacer region between them; and

d) The chimeric DNA molecule, when provided with plant cells, expresses the gene encoding an enzyme from the lactonase peptide biosynthetic pathway.

8. The chimeric DNA molecule of claim 7, characterized by the plant was selected from the group of Rutaceae Juss family, Aurantioideae sub-family, Citreae and Ciauseneae tribes, and subtribes comprising genera Citrus sp., Poncirus sp,, Fortunella sp., Murraya sp., Severinia sp. Microcitrus sp., Atalantia sp., and hybrids thereof.

9. Agrohacterium tumefaciens characterized by comprising the construct pBI-AI containing the aiiA gene, , as shown in SEQ ID n° 1 , that codes for an AHL lactonase enzyme, the nopaline synthetase ( OS) terminator and a gene conferring resistance to kanarnycin (NPTH), under control of the CaMV35S promoter, as shown in SEQ iD n° 2.

10. A transgenic plant characterized by comprising the aiiA gene encoding the AHL-lactonase enzyme that breaks down the signals produced by pathogenic bacteria, mitigating the Quorum sensing system thereof.

1 1. The transgenic plant of claim 10, characterized by being selected from the groups of Rutaceae Juss family, Aurantioideae sub-family, Citreae and Ciauseneae tribes, and subtribes comprising genera Citrus sp., Poncirus sp,, Fortunella sp., Murraya sp., Severinia sp. Microcitrus sp., Atalantia sp., and hybrids thereof.

12. The transgenic plant of claim 10, characterized by being transformed with a nucleic acid molecule that expresses the lactonase peptide, wherein the expression of the said peptide provides resistance to a bacterial pathogen having the Quorum sensing mechanism.

13. The transgenic plant of claim 10, characterized by having a phenotype of resistance to diseases having the Quorum sensing system, such as HLB (Huanglongbing) and other diseases with the same system.

14. A progeny characterized by being from the transgenic plant of claim 10.

15. A rootstock characterized by being from the transgenic plant of claim 10.

16. A somatic embryo characterized by being from the transgenic plant of claim 10.

17. A radicular, apical and side meristem, bud and budwood characterized by being from the transgenic plant of claim 10.

18. A ceii characterized b being from the transgenic plant of claim

10.

19. A seed characterized by being from the transgenic plant of claim

10.