Introduction to PR1 gene
PR 1 genes are responsible for the secretion of pathogenesis related (PR) proteins. These proteins work as the defence factors which are synthesized by the plants ubiquitouslyin response to the infections caused by the pathogens. They are produced by the plant cells after the pathogen derived molecules are recognized by the host plant cells and the transduction pathways are activated(Academic Press, 2014). They work in collaboration with the chitinases, glucanases and other phytoalexin biosynthetic enzymesand contribute indirectly or directly to the resistance from the pathogen attacks(Boller & Meins, 2012). The PR1 gene is synthesized in response to the infection, especially as a local hypersensitive response (HR) response to the virulent strain of the pathogen. The accumulation of PR 1 proteins in extracellular spaces also causes changes in the uninfected plant tissues which can lead to systemic acquired resistance (SAR)to the non-related virulent pathogens after the growth of HR. The signalling pathways of the PR genes are well understood and salicylic acid is the major component affecting the expression of this gene(Rivi?re, Marais, Ponchet, Willats, & Galiana, 2008).
Characteristics of genes
Primary source of gene: Araport:AT2G14610
Organism: Arabidopsis thaliana (ecotype: Columbia)
The gene expression is induced from a variety of organisms and is considered to be the molecular marker for SAR response. The cDNA which is associated to this gene is 'PR-1-like' and its gene expression is salicylic-acid responsive(National Center for Biotechnology Information, 2018).
The PR 1 proteins occur both in the acidic and basic isoforms and have no consistent amino acid sequence. The acidic PR 1 isoform is found in the extracellular spaces, vacuoles of idioblasts and xylem elements, whereas, the basic PR 1 isoform is found in mainly in the vacuoles of Nicotiana tobacum(UniProt Consortium, 2018).
The PR 1 genes are induced through signal transduction pathways which involve jasmonic acid, SA and ethylene. The pathwayof the acidic PR 1 genes was found to be induced through an SA-dependent pathway, whereas, the signalling pathway of basic PR 1 pathways was found to be induced using ethylene-dependent pathwayor JA-dependent pathway(Rivi?re, Marais, Ponchet, Willats, & Galiana, 2008).
Mode of action
The mode of action of this gene and its proteins is largely unknown and they are not associated with any enzymatic protein activity. The PR1 genes are commonly found in the angiosperms and apart from this, they area also found in the insects, yeasts and other vertebrates(Pirone & Shaw, 2012). However, the functions of the PR1 genes have been studied using the sequence comparison with the P25TI, a human protein gene which is homologous to the PR 1 gene of plants. The gene is related to the salicylic acid and when the plant is affected by a pathogen, the amount of salicylic acid increases in the affected site which induces the defence mechanism in the plants(Anderson, et al., 2011).
Why is banana plant chosen for research?
The reason behind choosing this for the research is that the Grand Naine variety of banana is the fourth most cultivated crop across the world. It is also valued all across the world as a staple crop. The plant lacks thegenetic diversity and thus, the unwanted experimental variables are eliminated and the validity of the results is increased.This is another huge reason for choosing the banana plant for this research. The elimination of the genetic diversity decreases the chances of unexpected expression of gene and the gene can be expressed in the way it is expected to. The researchers all across the world are trying to conduct the researches on the Grand Naine for creating more products of economic importance(Stover, 1987). Another reason which promotes the usage of this plant in research is the ability of this plant to multiply rapidly. The rapid multiplication reduces the time of results for the plant. The generation of plants with desirable gene expression can be attained in a few months. The plant can also be grown rapidly using tissue culture with less difficulty level (Kumar, 2016).
Meloigodyne incognita and its infection in Grand Naine
Meloigodyne incognita is a nematode which is considered to be one of the major obligate parasites of the agricultural crops including Grand Naine. This nematode species feeds on the root systems of the plant immediately after the infestation. The infestation of this species triggers a series of the physiological and morphological disruption of host which eventually leads to pathogenesis (Mohandas & Ravishankar, 2016). The reason for this disruption is the altered expression of gene in the affected cells and these disruptions can only be detected the molecular biology techniques (Ferji, Mayad, & Alfalah, 2013).
When the nematode species affects the Grand Naine, a reprogramming process in the plant metabolism takes place. The effector molecules of the nematode species showcase molecular manipulative and mimicry behaviour and reprogram the cells of the plant cells to form the feeding cells which suppress the defence responses of the plant(Pinochet, Fern?ndez, Jaizme, & Tenoury, 1997).
Vega and colleagues in 1997 conduct the study on the pathways adopted by the Grand Naine in overcoming the infection of the Meloidogyne incognita species on banana plant and found out that if the banana plants are inoculated with G. Mosseae, the plant nutrition in the banana plant is enhanced. This enhancement helps in the suppressing the reproduction and gall formation of the nematode species during the early stages of thedevelopment of the Grand Naine(Jaizme-Vega, Tenoury, Pinochet, & Jaumot, 1997).
To improve the defence mechanism and counter this infection, the plant cells express the PR1 genes which use the two different mechanisms. One such mechanism involves trans-membrane Pattern Recognition Receptor (PRR) and the intracellular immunity receptors which are known as Resistance (R) proteins. The PRR method responds to the infection through the molecular patterns such as PAMPs and MAMPs which induce the PAMP-triggered immunity (PTI)(Al-Idrus, Carpentier, Ahmad, Panis, & Mohamed, 2017). The method involving the resistance proteins, counters the effector molecules using specific R genes in plants which trigger a cascade of defence mechanisms. The defence mechanisms involve the stimulation of the hypersensitive responses (HR)in the cells which are located adjacent to the site of infection. After the stimulation of the HR, the SAR pathway of the banana cv. plant is also activated the HR pathway is stimulated as a result of oxidative burst which is signalled using calcium influx(Genoud, Buchala, Chua, & M?traux, 2002).
After the infection occurred in the plant, three KEGG pathways are witnessed in the plants which are related to maintenance of the giant cells and defence mechanism of the banana plant including the energy metabolism and lignification. The three KEGG pathways are namely glycolysis pathway, citrate cycle pathway and phenylpropanoid biosynthesis pathway(Mirzaei & Carrasco, 2016). The following images depict the three biosynthesis pathways:
Use ofPGEM T(VECTOR)
The pGEM T vector will be used for this research as it is a highly efficient TA cloning vector. It contains multiple cloning sites. The size of this vector is 3.0 kb and the ampicillin resistance site is present in it for gene selection. The ORF cDNA sequence of this vector can be amplified using the PCR with the RV-M primers and M13-47(Eun, 1996).
This vector is considered to be an easy vector and hence, is used by a number of researchers for cloning their genes and expressing them into different organisms. The vector has a series of ‘A’ and ‘T’ overhangs. In the PCR reaction, the Taq polymerase enzyme leaves A and T at the end of the amplified DNA(Chen & Janes, 2002). This makes the product of PCR to easily ligate with the vector and makes the gene transfer convenient. This vector also contains a variety of restriction sites which can be easily cut through endonucleases to create stuck ends(Elsevier, 2000).
The introduction of the PR1 gene in the banana cv. plant may help in increasing the defence mechanism of the plant against the nematode species Meloigodyne incognita by inducing the signalling pathways. This can help in eliminating the loss of crop encountered due to this parasite and hence, the economic value of the plant can be enhanced. Since, banana is the most recognized staple crop in many parts of the world and is considered to be the ultimate source of carbohydrates, any measure which can help in reducing the rate of deterioration or failure of this crop, will prove to be a boon for mankind.
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