Research Proposal: PR1 Gene in Banana during Nematode Infection

This research proposal focuses on the isolation and characterization of the PR1 gene in the Grand Naine variety of banana in response to infection by the nematode Meloidogyne incognita. The study explores the role of PR1 genes in plant defense mechanisms, specifically the production of pathogenesis-related proteins. The introduction highlights the importance of the banana crop and the impact of nematode infections. The proposal outlines the characteristics of the PR1 gene, its mode of action, and the signaling pathways involved, particularly salicylic acid. It justifies the choice of the Grand Naine banana due to its lack of genetic diversity and rapid multiplication rate. The proposal details the infection process of M. incognita and the plant's defense responses, including the activation of PR1 genes and the use of both PRR and R protein-mediated immunity. It includes the use of the pGEM T vector for cloning and emphasizes the potential for enhancing the plant's defense mechanism. The study aims to contribute to strategies that reduce crop loss and increase the economic value of bananas, a staple food source globally. References to relevant research papers and resources are included to support the proposed research.

Isolation and characterization of PR1
gene in Grand Naine (banana) during
Meloigodyne incognita infection
Research proposal

Table of Contents
Introduction to PR1 gene...........................................................................................................3
Characteristics of genes..........................................................................................................3
Mode of action.......................................................................................................................4
Why is banana plant chosen for research?.................................................................................4
Meloigodyne incognita and its infection in Grand Naine..........................................................5
Signalling pathways...................................................................................................................6
Use ofPGEM T(VECTOR)......................................................................................................10
Conclusion................................................................................................................................12
References................................................................................................................................12

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(Rivière, 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(Rivière,
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, Fernández, 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).

Signalling pathways
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, & Métraux, 2002).

Figure 1: Schematic defence pathway for compatible interaction between M. incognita and
Grand Naine
Source: (Al-Idrus, Carpentier, Ahmad, Panis, & Mohamed, 2017)
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:

Figure 2: Phenylpropanoid pathway
Source: (Al-Idrus, Carpentier, Ahmad, Panis, & Mohamed, 2017)

Figure 3: Glycolysis pathway
Source: (Al-Idrus, Carpentier, Ahmad, Panis, & Mohamed, 2017)

Figure 4: Citrate Acid cycle pathway
Source: (Al-Idrus, Carpentier, Ahmad, Panis, & Mohamed, 2017)
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).

Figure 5: The physical map of the pGEM vector
Source: ( Sino Biological Inc., 2018)
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).

Conclusion
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.
References
Sino Biological Inc. (2018). pGEM-T Vector. Retrieved from www.sinobiological.com:
https://www.sinobiological.com/Vector-pGEM-T-a-1636.html
Academic Press. (2014). Signaling Pathways in Plants. Academic Press.
Al-Idrus, A., Carpentier, S. C., Ahmad, M. T., Panis, B., & Mohamed, Z. (2017). Elucidation
of the compatible interaction between banana and Meloidogyne incognita via high-
throughput proteome profiling. PLOS OPne, 12(6).
Anderson, L. E., Aryamanesh, N., Shearer, B., McComb, J., Hardy, G. E., & O’Brien, P. A.
(2011). Phosphite primed defence responses and enhanced expression of defence
genes in Arabidopsis thaliana infected with Phytophthora cinnamomi. Plant
Pathology, 60(6).
Boller, T., & Meins, F. (2012). Genes Involved in Plant Defense. Springer Science &
Business Media.
Chen, B.-Y., & Janes, H. W. (2002). PCR Cloning Protocols. Springer Science & Business
Media.
Elsevier. (2000). RNA-Ligand Interactions, Part B: Molecular Biology Methods. Elsevier.