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General defense mechanisms in plants
Pathogen defense in plants has been extensively reviewed (Bent and Mackey, 2007). It begins with effective recognition of pathogen structures, or their effectors, by the plants that then initiate an appropriate defense response. Specialized cell-surface pattern recognition receptors (PRRs) in plants, such as flagellin sensing (FLS2), detect the conserved microbe associated molecular patterns (MAMPs) (Jones and Takemoto, 2004). This includes for example the bacterial cold shock protein (CSP), bacterial flagellin (a structural protein in bacterial flagella), bacterial elongation factor Tu (EFTu), bacterial lipopolysaccharides (LPS) and fungal chitin. On perception of a specific MAMP, signal-transduction cascades are activated culminating in establishment of basal defense mechanisms. This includes (i) callose and silicone deposition to reinforce the cell wall, (ii) closure of stomata, (iii) production of reactive oxygen species and (iv) transcriptional induction of pathogenesis-related genes (PR genes) (Nicaise et al., 2009).
Some plant bacterial pathogens can, however, suppress or evade these basal defense mechanisms. For example, Pseudomonas syringe secretes a virulence factor, termed coronatine (COR), which suppresses hormonal signaling. This phytotoxic molecule mimics the plant hormone jasmonic acid (Melotto et al., 2006) which suppresses stomatal closure via the inhibition of MAMP-induced abscisic acid (ABA) signaling in the guard cell. Coronatine also induces re-opening of stomata and multiplication of bacteria in both local and systemic tissues by inhibiting the accumulation of the key plant immune signaling molecule, salicylic acid (Zheng et al., 2012). Other bacteria, such as Agrobacterium tumefaciens and Xanthomonas campestris pv campestris, modify their flagellin (a MAMP) to avoid detection by FLS2 (a PRR). Other bacterial pathogens can further suppress basal defense mechanisms by directly injecting their effectors into host cells using a type three secretion system ( TTSS; a needle like pilus that injects effectors). For example, the TTSS effector protein, AvrPto from P. syringae pv. tomato blocks callose deposition in tomato plants. Similarly in Arabidopsis, the TTSS effector proteins AvrRpt2 from P. syringae pv. tomato and AvrRpm1 from P. syringae pv. maculicola, inhibits defense signaling induced by FLS2 pattern recognition receptors (Kim et al., 2005).
Some plant pathogenic bacteria can evade, suppress or shutdown basal plant defense mechanisms using T3SS effectors, but some plants use phenolic compounds to suppress this secretion system (Li et al., 2009). Other plants have also evolved mechanisms for directly or indirectly recognizing the presence of the effectors inside their cells. They use „disease-resistance‟ (R) proteins (Ade et al., 2007; Axtell and Staskawicz, 2003; Deslandes et al., 2003; Kim et al., 2002; Mackey et al., 2002). The R proteins act as specific intracellular receptors for the specific effectors and this sets off a cascade of defense responses rendering the host resistant (Gu et al., 2005a). Programmed and localized death of plant cells surrounding the area of attack, also referred to as the hypersensitive response (HR), is a mechanism of R protein-mediated defense response. This response is also associated with production of reactive oxygen species and nitric oxide. They appear to function both as signaling agents and as direct antimicrobial agents.
Recognition events in the plant defense response
Following pathogen recognition, there is an increased production of superoxide (O-2) on the apoplastic face of the plasmalemma at the site of infection and later in systemic tissues (Durrant and Dong, 2004). Superoxide dismutase (SOD) in the apoplast rapidly converts O-2 to H2O2. This may not only affect the pathogen, but also damages the plant cells when in high amounts. H2O2 will induce crosslinking at the plant cell wall thereby hindering pathogen penetration. It may also, especially at the site of attack, cause sudden death of plant cells thereby affecting the pathogen therein. This damaging effect must be controlled through detoxifcation of the accumulating H2O2. While H2O2 detoxification in plants may be achieved through the catalase-based mode, detoxifcation via peroxidases is more important as peroxidases are present in all cells and have a higher affinity for H2O2. This, however, requires a reductant. In plants the most important reducing substrate is ascorbate, which detoxifies H2O2 through the ascorbate-glutathione cycle (Foyer and Noctor, 2011; Noctor and Foyer, 1998). The enzyme ascorbate peroxidase uses electrons from ascobate to degrade H2O2 into water and monodehydroascorbate. Highly unstable monodehydroascorbate quickly dessociates into dehydroascorbate. This is later reduced to ascobate using electrones donated by glutathione, which becomes oxdised into the glutathione disufide form. Glutathione is later regenerated from the oxdised form in a reaction catalysed by glutathione reductase and NADPH. These changes in the redox state of cells causes monomerization of the cytosolic non-expressor of pathogenesis related protein1 (NPR1). This subsequently leads to NPR1 translocation into the nucleus and by interacting with TGA1 transcription factors causes pathogenesis-related (PR) gene expression (Foyer and Noctor, 2011; Mou et al., 2003). Resulting PR proteins, encoded by PR genes, are antimicrobial in nature with many of them having chitnase and glucanase activities.
Justification of study
The importance of banana Xanthomonas wilt is indicated by its wide distribution in Eastern Africa, the rapidity of its spread, its ability to cause total yield loss and threatening livelihoods of over 80 million people in the region. This study was aimed to identify a possible mechanism of resistance by comparing the responses of M. balbisiana, which is resistant, and the East African Highland Banana (EAHB) cultivar „Nakinyika‟ (AAAEA genome), which is susceptible to Xvm infection. The rationale for the selection of „Nakinyika‟ for this study was based on the finding that during the first phase of a current ABSPII project my institute successfully developed embryogenic cell suspensions (ECS) from several EAHB. An Agrobacterium-mediated gene transformation protocol was established to genetically transform the EAHB ECS. Successful transformation was achieved for three EAHB cultivars (‘Nakinyika’, ‘Nakasabira’ and ‘Mpologoma’). Accordingly, any identified resistance mechanism in M. balbisiana could potentially be exploited and supplement ongoing genetic engineering research by my research group to generate resistant (EAHB) banana. Various plant defense pathways were therefore explored in this study both at morphological and molecular level in order to identify any trait that confired Xvm resistance to M. balbisiana. ‘Nakinyika’ is a typical cooking variety cultivar that is an important staple in East and Central Africa, which is often eaten after steaming. While specific information on the overall nutritional content of its edible pulp is scanty, available data shows that it is an important source of vitamin A, at 67.6 μg/100 g retinol equivalent (Bresnahan et al., 2012). At this level, one needs to consume 4.4 kg of pulp to attain 3000 μg as daily requirement for an average person. This value from ‘Nakinyika’ is only inferior to a few varieties among the EAHB including „Butobe’ (169.2 μg/100 g), „Nakitembe’ (88.03 μg/100 g), „Entukura’ (81.86 μg/100 g), „Nakhaki’ (77.17 μg/100 g) and „Kibuzi’ (71.62 μg/100 g) (Fungo and Pillay, 2011). Studies in my laboratory have shown that ‘Nakinyika’ is also an important source of iron and zinc at 9 mg/kg and 8 mg/kg of dry weight of pulp, respectively. In comparison to other varieties commonly consumed among the EAHB, its iron content is superior to that of Nakitembe (7 mg/kg), another popular cooking EAHB variety. Its iron content is also within range of other popular EAHB cooking varieties of „Mbwazirume’ and „Mpologoma’ at 10 mg/kg but inferior to that of „Kisansa’ (11 mg/kg), another popular EAHB variety. The recommended dietary allowance for iron for an average adult male is 8 mg while that of the female is 18 mg. This means that if ‘Nakinyika’ was the sole source of iron, an adult male would need to consume one kg while a female would need 2.5 kg per day. The zinc content of ‘Nakinyika’ on the other hand is similar to that of the other popular EAHB varieties of „Kisansa’ and „Nakitembe’ (7-10 mg/kg), „Mbwazirume’ and „Mpologoma’ (8 mg/kg). The recommended dietary allowance for zinc for an average adult male is 11 mg while that of the female is 8 mg. This means that if ‘Nakinyika’ was the sole source of zinc, an adult male would need to consume 1.5 kg while a female would need 1kg per day.
In general, M. balbisiana is a wild banana native to eastern South Asia, northern Southeast Asia, and southern China. It is characteristically with lush leaves in clumps with a more upright habit than most cultivated bananas. Further, M. balbisiana accessions have been previously found to be resistant against Xvm in a greenhouse trial (Tripathi and Tripathi, 2009). Also in screen-house and field trials, this type of banana developed no symptoms over 6 weeks after a single inoculation at dosages that caused the disease on a control (Kumakech et al., 2013). Only after a second inoculation at 6 weeks did any disease symptoms develop.
CHAPTER ONE
GENERAL INTRODUCTION AND LITERATURE REVIEW
1.1 Description and characterization of bananas and plantains
1.2 Banana and plantain distribution and production trends
1.3 Economic importance of banana
1.4 Major constraints to banana and plantain production
1.5 Mechanisms of resistance
1.6 General defense mechanisms in plants
1.7 Recognition events in the plant defense response
1.8 Justification of study
1.9 Working hypothesis and objectives
CHAPTER TWO
DEVELOPMENT OF A PROTOCOL FOR IN-VITRO GENERATION AND ALSO REGENERATION M. BALBISIANA AND EAHB CV. „NAKINYIKA‟ PLANTLETS BY TISSUE CULTURE
2.1 Abstract
2.2 Introduction
2.3 Materials and methods
2.4 Results
2.5 Discussion
CHAPTER THREE
CHARACTERIZATION OF THE MORPHOLOGICAL RESPONSE OF MUSA BALBISIANAAND EAHB CV „NAKINYIKA‟ TO BANANA XANTHOMONAS WILT INFECTION
3.1 Abstract
3.2 Introduction
3.3 Materials and methods
3.4 Results
3.5 Discussion
CHAPTER FOUR
MOLECULAR RESPONSE AND PHENOLICS PRODUCTION OF MUSA BALBISIANA
4.1 Abstract
4.2 Introduction
4.3 Materials and methods
4.4 Results
4.4 Discussion
CHAPTER FIVE
GENERAL DISCUSSION
References
