The modification of JA by jasmonate carboxyl methyltransferase converts it into the volatile compound methyl JA . This reaction is presumed to take place in the cytoplasm . MeJA mediates both developmental processes and defense responses against biotic and abiotic stresses . Additionaly, MeJA is a gaseous compound, and can thus act as airborne signals to mediate inter-plant communication. This means that neighboring plants can be signaled if stressed are present . Research has indicated that a positive feedback loop exists for JA biosynthesis . In Arabidopsis, VSP expression is selectively induced by JA and not OPDA, while both molecules induce defense responses. This demonstrates that multiple mechanisms are responsible for the transduction of JA-related signals, which are selectively activated in response to differing stimuli . The bacterial plant pathogen Pseudomonas syringae produces a JA-Ile-like compound termed coronatine , which suppresses some SA-immune responses by activating the JA signaling pathway . The Arabidopsis mutant coronatine-insensitive 1 was identified due to its insensitivity to plant growth inhibition by COR and OPDA . In addition, this mutant is male sterile and more susceptible to some pathogens and pests . COI1 encodes a 66-kD protein that contains an N-terminal F-box motif and a leucine-rich repeat domain . F-box proteins recruit regulatory proteins as substrates for ubiquitin-mediated degradation. This requires them to associate with Skp1 and Cullin to form an E3 ubiquitin ligase termed SCF complex which is important for JA responses .
JA–Ile facilitates binding of SCFCOI1 with the jasmonate ZIM-domain 1 protein . JAZ proteins are substrates for SCFCOI1 and negatively regulate JAresponses . In Arabidopsis these proteins belong to a 12 member family and contain a conserved motif at the C-terminus . Many JAZ mutants displayed no discernible phenotypes,vertical rack system possibly due to functional redundancy . Mutants of jaz10 are hypersensitive toJA, but the lack of obvious phenotypes in other jaz mutants suggests functional redundancy among other family members . The jaz1 mutant on ther other hand displayed male sterility, JA insensitivity, and increased resistance to infection by the bacterial pathogen P. syringae pv. tomato . The JAZ proteins homo- and hetero-dimerize by way of a conserved TIFY domain and also bind AtMYC2 and interact with COI1 through their C-terminal JAS domains . AtMYC2 is a helix-loop-helix transcription factor that acts as a key regulator of JA responses in plant-microbe interactions . When JA-Ile or COR bind to SCFCOI1 complexes, this promotes the ubiquitination of JAZ proteins leading to their degradation. Once degraded the JAZ-dependent repression on AtMYC2 is relieved and JA-responsive genes are activated . The JAZ proteins then recruit a co-repressor TOPLESS 8 and TPL related proteins through a adaptor protein called Novel Interactor of JAZ . NINJA and TPL proteins act as negative regulators of jasmonate responses . Resistance to specific pathogens conferred through JA signaling show little overlap in transcriptional changes. Molecular recognition of necrotrophic pathogens can trigger increased JA and ET synthesis as well as expression of defense genes, such as PDF1.2. Defensins, one group of ubiquitous peptides involved in innate immune response, are found in organisms ranging from invertebrates to plants. They are small , basic, cysteine-rich proteins encoded by multigene families, similar in complexity to those encoding other defense-related proteins . Defensins were first discovered in rabbits in 1984 and described in wheat and barley in 1990 .
PDF genes were later discovered in Arabidopsis and encode small peptides originally labeled as γ-thionins . γ-thionins were later renamed plant defensins to emphasize their structural similarity to mammalian defensins. The ancestry of defensins is thought to pre-date the divergence of plants and animals . Defensins are described in two separate kingdoms and further divided into five classes. Class 1, Class 2 and Class 3 are found in mammals and birds, while Classes 4 and are found in insects and Class 5 in plants . The primary structures of defensins vary between organisms. PDF proteins contain eight conserved cysteines . They also include a common fold, which is formed by a β-sheet and an α-helix steadied by disulfide bridges and capable of stabilizing the entire protein . Arabidopsis PDF genes can be separated into three families, each encoding closely related peptides. Family 1 consists of PDF1.1-1.5, family 2 consists of PDF2.1–2.6, and family 3 consists of At4g30070 and At5g38330 . The predicted mature peptide sequences encoded by PDF1.2a/b/c are identical. PDF1.2aand PDF1.2c form a tandem repeat on chromosome 5 and PDF1.2b is found on chromosome 2 directly adjacent to PDF1.3 . The high sequence similarity between PDF1.2a/b/c and PDF1.3indicates that little evolutionary time has passed since their duplication. More amino acid variability is evident within the second family but this family also has genes that occur in a tandem array . PDF2.1 is expressed specifically in roots, siliques, and seeds . PDF2.2 is expressed in all organs of healthy plants except stems and seeds . PDF2.3 is expressed in all organs excluding roots and is not upregulated in response to pathogen infection . PDF1.2a is induced in leaves upon infection of pathogens such as Botrytis cinerea . PDF1.1 expression is largely seed specific and may protect seedlings against pathogens . PDF1.1, 1.2, 2.1, 2.2 and 2.3 display distinct organ-specific expression patterns . PDF1.1, PDF2.1, PDF2.2 and PDF2.3 are expressed constitutively which means that most plant tissues constitutively express two or more defensin genes at any given time . Therefore, it is likely that specific peptides may be expressed during specific situations and sites . PDF1.4 and PDF2.4 appear not to contain predicted signal peptide sequences, suggesting they stay in the cytoplasm while the others PDF proteins are secreted.
Alternately, PDF proteins may overlap while acting distinctly to cover gaps in each-others’ activity spectrum . From the third family found within Arabidopsis, PDF3.2 encodes a protein of 129 amino acids with a C-terminal domain that has the conserved cysteine pattern shared by all plant defensins . PDF3.1 encodes 122 amino acids with 56% identical residues to the protein encoded by PDF3.2. These proteins could be fusion proteins or precursors but more research is necessary to determine their functions . Transgenic expression of PDF genes leads to fortification of tissues against pathogen attack. Two PDF proteins, one from dahlia and one from radish were found to inhibit the growth of Neurospora crassa. One observed response after infection with N. crassa is a change in fungal cell ion fluxes,mobile grow rack but this is believed to be a secondary effect, not a direct result of the PDF anti-fungal activity . There are two models describing possible modes-of action for the antimicrobial activity of PDF proteins. One model proposes that pores form in microbial cell membranes. The second model proposes that PDF proteins bind onto the anionic lipid head groups disrupting the stability of the phospholipid bilayer of microbial cell membranes . Binding sites for these PDF proteins to sphingolipids found on the cell surface in fungal membranes have been demonstrated . This suggests that the antimicrobial activity of PDF proteins is dependent on their ability to bind to a target in the membrane of an infecting pathogen . No similar activity has been shown against pathogenic fungi though. Aside from the many possible roles of PDF genes it has been established that many PDF proteins isolated to date display antifungal activities against a broad range of fungi. Monocot PDF proteins may also inhibit α-amylase, an enzyme found in insect guts . This possibly hampers the insect’s ability to digest plant material . It appears though while some alternately inhibit α-amylase activity and protein synthesis that PDF proteins do not display both activities concurrently. Additionally, most plant defensins do not display antibacterial activity which has been suggested to be a result of a infection pressure from fungal as opposed to bacterial pathogens on plants . Additional biological activities of PDF proteins include the ability to inhibit protein translation in a cell free system, inhibit proteases, and inhibit the growth of microbes . They may also act as mediators of zinc tolerance and inhibitors of ion channels, or exhibit activity against mammalian cells and enzymatic activity controlling the redox state of ascorbic acid . This diverse set of functions may be attributed to differences in the primary structures of defensins. PDF activities range from antimicrobial and insecticidal to anti-parasitic.
Due to their broad range of target organisms, PDF overexpression may be a suitable strategy for crop protection. While R-genes typically confer a narrow range of resistance, PDF genes may provide broad-spectrum resistance against multiple types of pathogens. If introduced into plants PDF genes may offer defensive advantages in addition to enhancing the plants ability to combat biotic stressors . Arabidopsis also contains over 300 Defensin-like genes and the evolution of DEFL-, PDF-, and R-genes bears remarkable similarity that may be due to their evolution through similar mechanisms . Members of these three groups can be found as single genes or in clusters, which arose through duplication, recombination, or diversifying selection . Cis-elements discovered within the promoter of PDF1.2b include a GCC box important for the ET response, a stress responsive G-box, a drought responsive element , and JA-responsive elements and . GCC boxes are commonly found in the promoters of genes which encode defenserelated proteins and are known binding sites for some AP2/ERF transcription factors . G-boxes regulate genes in response to environmental conditions such as red and UV light, anaerobiosis, and wounding and can be bound by certainbasic leucine zipper transcription factors . Transcription factors that positively affect PDF1.2b mRNA levels are the AP2/ERF-domain transcription factors ERF1, ORA59, AtERF1 and AtERF2, as well as the basic leucine zipper transcription factor TGA5 . Negative effects were observed upon over expression of AtERF4 or WRKY70, as well as in a triple mutant, where function of TGA2, TGA5 and TGA6, three redundantly acting TGA bZIP factors, is abolished . TGA2, TGA5 and TGA6 are also commonly required for the activation of JA- and ET-dependent defense mechanisms that counteract necrotrophic pathogens . TGA2 is the only transcription factor, for which direct interaction with the PDF1.2 promoter has been demonstrated yet . ORA59 and ERF1 are believed to bind directly to the promoter and AtERF1 and AtERF2 indirectly . ORA59 is the primary positive regulator of PDF1.2b expression in response to JA/ET and it binds GCC boxes . Since most PDF genes belong to small families of closely related members, studying their function can be difficult using traditional genetic methods. Hpa is a useful model pathogen to study the genetics of plant-pathogen interactions . However, unlike infections with bacterial pathogens, inoculation with Hpa does not result in synchronous and uniform responses in host plants. This is mainly due to the unsynchronized germination of Hpa spores upon spray inoculation. Furthermore, transcription factors and other regulators controlling defense responses are frequently represented by families of functionally redundant members . Additionally, some components of the plant immune system may be essential for plant survival during early development . Hence, conventional genetics is of limited use for the dissection of the plant defense network in this circumstance. Chemical genetics utilizes small molecules to alter in vivo protein functions in a reversible and highly controllable manner . Using such bioactive compounds as synthetic elicitors, a simultaneous and uniform activation of the defense network can be accomplished . This allows for reproducible measurements of the dynamics of molecular processes and physiological responses. In addition, synthetic elicitors may be used to simultaneously knock out families of functionally redundant proteins, theoretically resulting in clear phenotypes. Moreover, the function of essential genes can be studied efficiently by introducing the respective chemical at any stage of development to manipulate their activity . The identification of a collection of novel synthetic elicitors may permit the selective manipulation of defined branches of the defense network. Such elicitors will serve as powerful new tools for the emerging field of systems biology. They are likely to facilitate the stimulation of the defense network with unprecedented precision allowing the examination of the relation of defined signaling events and physiological outputs in a quantitative manner. In the broader context, the ability to manipulate disease resistance pathways using synthetic elicitors can also be exploited for agricultural purposes by using various combinations of chemicals to develop a new generation of pesticides.