Plants resistant to pathogenic microorganisms growing in vascular tissues

A microbial and antimicrobial technology, applied in the direction of plant peptides, plant products, botany equipment and methods, etc., can solve problems such as no effective control and/or coping methods

Active Publication Date: 2016-03-16
CENT DE INVESTIGACION & DE ESTUDIOS AVANZADOS DEL INST POLITECNICO NACIONAL +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0038] Notwithstanding the above, it is necessary to propose methods and / or effective control strategies to address plant diseases caused by microorganisms that invade the phloem via symplasts, because prior to the present invention, there were no effective control and / or countermeasures

Method used

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  • Plants resistant to pathogenic microorganisms growing in vascular tissues
  • Plants resistant to pathogenic microorganisms growing in vascular tissues
  • Plants resistant to pathogenic microorganisms growing in vascular tissues

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0125] Example 1. Obtaining the CsPP16 expression unit comprising the 35S promoter of cauliflower mosaic virus

[0126] To obtain expression units with the sequences listed in Table 4, they were synthesized and assembled in the order listed; they were sequenced to verify the absence of insertional mutations during assembly, and finally cloned into cells containing carbenicillin antibiotic resistance. Genscript vector.

[0127] The resulting recombinant plasmids were transformed into competent cells of A. tumefaciens and A. rhizogenes. We use the antibiotic carbenicillin, which is a more stable and synthetic form of penicillin. Plasmid DNA was extracted from resistant bacteria and the presence of the recombinant plasmid was verified on an agarose gel.

[0128] To verify the presence of the gene of interest with antimicrobial activity inserted into the vector or in plants transformed thereby, specific oligonucleotide primers are used for PCR amplification; for example, in th...

Embodiment 2

[0129] Example 2. Obtaining a CsPP16 expression unit comprising the vascular promoter AT5G59880

[0130] To obtain this expression unit, the shorter version of the promoter 35S of cauliflower mosaic virus (SEQ.ID.No. 2) or the longer form (SEQ.ID.No.7). As in Example 1, the sequences were synthesized and assembled in the order listed, they were sequenced to verify the absence of insertional mutations during assembly, and finally cloned into a Genscript vector containing carbenicillin antibiotic resistance.

[0131] As in Example 1, the resulting recombinant plasmids were transformed into competent cells of A. tumefaciens and A. rhizogenes. We use the antibiotic carbenicillin, which is a more stable and synthetic form of penicillin. Plasmid DNA was extracted from resistant bacteria and the presence of the recombinant plasmid was verified on an agarose gel.

[0132] Table 4

[0133]

[0134] Table 4 (continued)

[0135]

[0136] To verify the presence of the gene of...

Embodiment 3

[0137] Example 3. Transient expression of a construct of the invention in HLB-infected Mexican lime plants using Agrobacterium

[0138] Agrobacterium was grown in LB medium at 30°C under constant agitation to achieve an O.D. (600nm) of 0.4. Acetosyringone was added to the cells at a final concentration of 140 micromolar and incubated for an additional two hours under the inducer. Bacteria were harvested by centrifugation and resuspended in transformation medium ( Figure 7 ).

[0139] The method of inoculating adult plants involves removing the lignified bark with sandpaper, exposing the green photosynthetic tissue. Pre-wet swabs with recombinant bacteria containing some of the expression vectors described in Examples 1 and / 2 were placed on the tissue and resuspended in transformation medium. The swabs were temporarily fixed around photosynthetic tissue not covered with plastic, and the plants were covered with plastic bags and kept in high humidity for two days. Under inc...

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Abstract

The present invention relates to the generation of transgenic plants resistant to infections caused by microorganisms restricted to the phloem, and it comprises the induction of the expression of a chimeric or fusion protein, which comprises a phloem protein of Citrus aurantifolia (CsPP16), a linker protein and a protein with antimicrobial activity; for example, with antibacterial activity or antibacterial hydrolytic enzyme. The portion of the fusion protein corresponding to the CsPP16 protein can exert its function of simplasmic movement, which occurs via the plasmodesmata of the plant to reach the phloem sieve tubes, while the antimicrobial portion can act to eliminate the microorganism, which settles in the phloem. The invention allows controlling the disease that causes the citrus yellowing or Huanglongbing (HLB), also applying to the treatment of other diseases caused by bacteria and other microorganisms that settle in the vascular tissue of higher plants. The invention includes the designed molecular vectors carrying and expressing a fusion protein and the transgenic plants obtained with a stable expression of said fusion protein and the transformation method of adult plants, healthy or diseased, to express the chimeric proteins and controlling, e.g., HLB disease. The transformation of diseased plants with the fusion protein is a method to produce healthy shoots free from the microorganism causing the disease; e.g., free from HLB bacteria.

Description

technical field [0001] The field of the invention is the application of molecular biology techniques to produce phytotoxicity in the vascular tissues of tolerant plants by expressing proteins that have antimicrobial activity and are translationally fused to proteins capable of supracellular and systemic mobilization. Plants with pathogenic microorganisms. In one of its embodiments, the present invention relates to a tissue-specific vascular antimicrobial protein fused to a protein capable of simplasmic movement through vascular tissue to distal tissues by constitutively expressing Methods of controlling bacteria that cause citrus yellowing or Huanglongbing (HLB). Background technique [0002] Huanglongbing (Huanglong in Chinese) is a citrus disease of great importance in Mexico. It is also known by the initials HLB and the English words Greening or Ex-Greening. The disease is very destructive as it can cause total losses in citrus production. Symptoms of HLB include but ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A01H5/00C12N15/82
CPCC07K14/415C07K2319/00C07K2319/55C12N15/8281C12N15/8205
Inventor B·霍康诺斯托卡扎雷斯C·查瓦林帕拉西奥J·A·洛佩斯布埃菲尔M·A·格拉卢皮安O·莫拉莱斯加尔万R·泽库纳赫拉R·鲁伊斯梅德拉诺A·戈麦斯费利佩F·J·特鲁希略阿里亚加
Owner CENT DE INVESTIGACION & DE ESTUDIOS AVANZADOS DEL INST POLITECNICO NACIONAL
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