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Strategies for Producing Pathogen-resistant Plants






Natural resistance

A potential pathogen has to overcome sev- eral barriers in order to infect a plant and become an actual pathogen (Thordal- Christensen, 2003). These barriers can be pre-formed (wax, cell walls, secondary metabolites and antimicrobial enzymes), but are often active responses following the recognition of the pathogen (Thordal- Christensen, 2003). Plants often respond in similar ways to host and non-host pathogens sharing some of the same signal components (Thordal-Christensen, 2003). The outcome of an interaction with a pathogen is thus governed by many factors, including the genotypes of the plant and pathogen as well as a complex exchange of signals between the two (McDowell and Dangl, 2000). Activation of inducible defences is contingent upon recognition of an invasion. Structural molecules from the pathogen such as cell wall components and bacterial fl agellin elicit a defence response and are considered ‘general elicitors’ (Martin et al., 2003). However, a more spe- cific detection system by the plant is through a complex array of constitutively expressed R (for resistance) genes (Martin et al., 2003). Individual R genes have narrow recognition capabilities and they trigger resistance only when the invading pathogen expresses a corresponding Avr (for avirulence) gene. Avr proteins can specifi cally modify host targets that are detected by the R proteins in resistant plants (Zhu et al., 2004). R gene-mediated resistance (also called gene-for-gene resist- ance) is commonly, although not invari- ably, associated with rapid necrosis of plant cells at the site of invasion, the hypersensi- tive response, that stops pathogen infec- tion. Six distinct classes of highly polymorphic but structurally conserved R proteins that mediate resistance against dif- ferent pathogen taxa have been identifi ed (Lahaye, 2002). The majority of the cloned R genes encode putatively cytoplasmic pro-


 

 

teins with a nucleotide-binding site (NBS) and a C-terminal leucine-rich repeat (LRR) domain (Lahaye, 2002). NBS–LRR proteins are further divided into two subclasses depending on whether the N-terminal domain is a Toll/interleukin-1 receptor (TIR) or coiled-coil (CC) motif. The LRR backbone is proposed to provide a versatile recognition surface for specifi c Avr percep- tion, while evidence indicates that the TIR and CC domains mediate downstream sig- nalling (Lahaye, 2002).

Downstream of recognition, which occurs when plants first encounter pathogens, more global resistance mecha- nisms are induced. The earliest detectable cellular events are ion fl uxes across the plasma membrane and a burst of oxygen metabolism that produces reactive oxygen intermediates (ROIs), such as superoxide (O2–) and hydrogen peroxide (H2O2)

(McDowell and Dangl, 2000; Delledonne et

al., 2002). Receptor-mediated ion fl uxes trigger localized production of nitrous oxide (NO) and ROIs immediately after pathogen recognition. These second mes- sengers synergistically induce cell death, production of salicylic acid (SA), defence gene expression and more ROIs, establish- ing a putative feedback loop in which the response is amplifi ed. Characterization of these steps in plant defence is an area of very active research.

An important component of this defence system is systemic acquired resist- ance (SAR) (recently reviewed by Durrant and Dong, 2004). After a hypersensitive response to invading pathogens, plants show elevated accumulation of SA, induced expression of pathogenesis-related (PR) genes and SAR to further infection by a broad range of pathogens. Considerable effort has been directed toward identifying the signalling molecules responsible for activating the hypersensitive response and SAR, and there is now compelling evidence that SA plays a crucial role in triggering SAR. Application of SA or its analogues to both tobacco and Arabidopsis induces PR gene expression and resistance as would a biological agent (Shah et al., 1999).


 


Transgenic tobacco plants that express the bacterial salicylate hydroxylase (nahG) gene cannot accumulate SA or develop SAR, and exhibit heightened susceptibility to pathogen infection (Delaney et al., 1994). Likewise, preventing SA synthesis by specifi cally inhibiting the activity of pheny- lalanine ammonia-lyase, the fi rst enzyme in the SA biosynthetic pathway, makes other- wise resistant Arabidopsis plants suscepti- ble to Peronospora parasitica (Mauch-Mani et al., 1996). Further, at least some R genes are upregulated by SA, forming a feedback amplifi cation signal loop (Shirano et al., 2002; Xiao et al., 2003). Two distinct signal transduction pathways that conduct to SAR after pathogen recognition by R proteins have been identifi ed (Fig. 14.1). Two genes from Arabidopsis, EDS1 and PAD4, that encode lipase-like proteins and that inter- act with each other, mediate the down- stream signalling of TIR- but not CC-type R protein receptors. Further, EDS1 and PAD4 seem to be a converging point of TIR-medi- ated resistance to bacteria, fungi and viruses (Liu et al., 2002; Whitham et al., 2003). A membrane-associated protein encoded by NDR1 is required to trigger resistance by many CC R proteins, but not by TIR proteins. Another set of genes also required for signal transduction, RAR1, SGT1 and PBS3, overlap the pathways described above, indicating the complexity of the signal pathway. RAR1 is a compo- nent of the N-mediated resistance to Tobacco mosaic virus (TMV) and some pow- dery mildews; however, it is not required by all R genes (Liu et al., 2002; Quirino and Bent, 2003). SGT1 and RAR1 physically interact and they have been suggested to be associated with the ubiquitination and sub- sequent degradation of proteins (Martin et al., 2003; Quirino and Bent, 2003). Further, a molecular chaperone (Hsp90) interacts in Nicothiana tabacum with SGT1, RAR1 and the N protein, and is also required for the signal transduction of R gene- mediated resistance (Liu et al., 2004). This may indicate that some R proteins are able to assemble into recognition complexes without the help of chaperones, hence


explaining the different requirements of R genes for SGT1/RAR1 resistance (Liu et al., 2004).

In addition to SA, ethylene (ET) and jasmonic acid (JA) serve as important sig- nals for the induction of various defence responses, including insects, mechanical damage and necrotrophic pathogens; they work with SA to signal some but not all defence responses, and in some cases func- tion independently (Pieterse and van Loon, 1999; Shah et al., 1999). A different form of systemic resistance is the induced systemic resistance (ISR) that requires signal path- ways responding to the hormones ET and JA and is independent of SA. In nature, cer- tain non-pathogenic bacteria can trigger this response. Both SAR and ISR pathways are part of a complex signal network with members that seem to act independently but coordinately (Fig. 14.1). Overall, the SA and JA/ET pathways act in opposition to each other although some overlap exists (Glazebrook et al, 2003).

One of the key genes involved in both SAR and ISR is the Arabidopsis thaliana NPR1/NIM1 that functions as a signal mod- ulator (Fig. 14.1) (Cao et al., 1997, 1998; Yu et al., 2001). Upon induction (by pathogen infection and/or SA), NPR1 expression is elevated and the NPR1 protein is activated, in turn inducing expression of a battery of downstream PR genes. The NPR1 gene encodes a protein containing ankyrin repeat motifs of the sort mediating pro- tein–protein interactions in animals (Cao et al., 1997). Arabidopsis mutants lacking a functional NPR1 gene are unable to express PR genes in response to SA or its analogues. In addition, npr1 mutant plants show increased susceptibility to fungal, bacterial and viral pathogens. The recessive nature of most identified NPR1 mutant alleles strongly suggests that NPR1 is a positive regulator of the SA signal transduction pathway. In uninduced cells, NPR1 exists as an oligomer formed by intermolecular disulphide bonds. After an oxidative burst during SAR induction and the subsequent accumulation of antioxidants, the cell becomes a more reductive environment and


 

 

Fig. 14.1. Systemic signalling in Arabidopsis disease resistance. Recognition of invading pathogens is medi- ated by resistance (R) proteins (CC– and TIR–NBS–LRR). Two distinct signal transduction pathways conduct to SAR. EDS1 and PAD4, that encode lipase-like proteins and interact with each other, mediate the down- stream signalling of TIR- but not CC-type R protein receptors. NDR1 is required to trigger resistance by many CC R proteins, but not by TIR proteins. RAR1 and SGT1 overlap the pathways described above. After induction, the levels of salicylic acid (SA) increase, the cell becomes a more reductive environment and NPR1 is converted from an oligomeric to a monomeric state. In this reduced state, NPR1 is translocated to the nucleus where it interacts with members of the basic leucine zipper (bZIP) family of transcription factors (TGA) to induce the activity of pathogenesis-related (PR) genes. Some non-pathogenic bacteria, necrotrophic pathogens, insects and wounding induce a separate defence pathway that is dependent on jasmonic acid (JA) and ethylene (ET). This pathway ultimately produces the induction of defensins (PDF1.2). SAR = systemic acquired resistance; ISR = systemic induced resistance. Solid arrows indicate a positive effect; dashed arrows indicate a partial requirement; and lines with a bar indicate an inhibitory effect.

 


NPR1 is converted to a monomeric state by the reduction of intermolecular disulphide bonds (Mou et al., 2003). In this reduced state, NPR1 is translocated to the nucleus where it interacts with members of the basic leucine zipper (bZIP) family of tran- scription factors (TGA factors) to induce the activity of PR genes (Kinkema et al., 2000; Fan and Dong, 2002; Johnson et al., 2003). Interestingly, one TGA factor (TGA1) also has to be reduced before it is able to inter- act with NPR1 (Despres et al., 2003).


Cytoplasmic NPR1 appears to modulate cross-talk between SA and JA defence path- ways (Spoel et al., 2003). Constitutive over- expression of NPR1 in Arabidopsis did not result in constitutive PR gene expression in the absence of pathogens; however, it did lead to enhanced resistance to the bac- terium Pseudomonas syringae and the oomycete Peronospora parasitica, with no obvious detrimental effect on the transgenic plants (Cao et al., 1998; Friedrich et al., 2001). NPR1 homologues have been identi-


 


fi ed in a variety of economically important plants, including rice, soybean and maize. The ubiquitous existence of NPR1 in differ- ent plant species suggests that the fi ndings with NPR1 in Arabidopsis are likely to apply to other species. Recently, the Arabidopsis NPR1 gene was overexpressed in rice plants and the transgenic plants showed enhanced resistance to Xanthomonas oryzae pv. oryzae (Chern et al., 2001), indicating conserved signal transduction pathways controlling NPR1- mediated resistance among widely diverged plant species (in this case dicots and mono- cots).

At least some early SA-activated genes (glutathione S -transferase, GST6 and glucosyltransferase, EIGT) do not require NPR1 for their induction. This points toward different mechanisms for early and late gene activation despite the fact that they require SA and TGA, and share some promoter elements with late acti- vated genes such as PR1 (Uquillas et al., 2004).

 

Protein kinases and signal transduction

One of the early cellular events that occur within minutes of pathogen recognition is the activation of protein kinases. The evi- dence suggests that they have an essential role in early signal transduction. Some of the rapidly activated kinases have been identified as mitogen-activated protein kinases (MAPKs) (Peck, 2003). MAPKs are activated by general elicitors such as fl a- gellin, chitin and other fungal cell wall components, as well as specifi c elicitors recognized by the R protein transduction pathway (Ekengren et al., 2003). This is an indication that kinases could be a converg- ing point for host and non-host signal trans- duction pathways. Further, at least in one system (Pseudomonas syringae pv. tomato carrying the AvrPto or AvrPtoB avirulence genes and tomato carrying the Pto resist- ance gene), two MAPKs (WIPK and NTF6) and two MAPK kinases (MEK1 and MEK2) are essential for the activation of the NPR1- mediated SA pathway (Ekengren et al.,


2003). On the other hand, EDR1 of Arabidopsis (a MAPK kinase kinase), is a negative regulator of the SA defence path- way (Frye et al., 2001). Orthologues of EDR1 are present in several species of dicots and monocots, indicating that it must be part of a conserved pathway in plants (Frye et al., 2001; Tang and Innes, 2002; Kim et al., 2003).

The questions we are asking in our research are: do these same genes and disease signal transduction pathways func- tion in the perennial plant citrus in the same manner as in annual plants? Can over- expression of genes in the pathways be used to provide generalized pathogen resistance? These issues are discussed fur- ther below.

 

 

Pathogen-derived resistance

In 1985, Sanford and Johnson advocated the use of pathogen-derived genes for gen- erating host resistance. Proof of concept was demonstrated shortly thereafter by Roger Beachy’s group, who showed that the expression of a viral coat protein (CP) gene in a transgenic plant could confer resist- ance to the donor (Powell-Abel et al., 1986). In the intervening years, there have been numerous reports of various virus–host combinations where this strategy has been successful to some degree.

Once this strategy was found to be suc- cessful, investigations into the underlying mechanism(s) of resistance began. It was found that there was indeed more than one mechanism operating and that they could be protein mediated or RNA mediated. The best studied example of protein-mediated resistance is that of TMV resistance con- ferred by expression of the TMV CP gene in transgenic tobacco by Beachy’s group as referenced above. In this case, it was found that virus resistance depends on the syn- thesis of transgene-encoded CP, since a non-coding TMV CP gene was not effective (Powell et al., 1990). The resistance can be overcome when plants are inoculated with uncapsidated viral RNA rather than virions,


 


 

leading to the conclusion that resistance is due to interference of the transgenic CP with virion disassembly (Nelson et al., 1997). In grafting experiments, the trans- genic CP also inhibited systemic spread of the virus (Wisniewski et al., 1990), which might implicate a second mechanism of protection. However, systemic movement of the TMV virions may also require virion disassembly and assembly (Baulcombe, 1996).

RNA-mediated mechanisms were also documented early on. In 1987, the year fol- lowing the fi rst description of resistance in CP-transgenic plants, the very fi rst virus- resistant transgenic plants in which resist- ance was clearly due to RNA-mediated phenomena were reported. In plants expressing a satellite RNA, either Cucumber mosaic virus (Harrison et al., 1987) or Tobacco ringspot virus (Gerlach et al., 1987), resistance was thought to be due to competition for replication between these non-coding RNAs and the viral genomic RNAs. The same mechanism has been invoked more recently when other non- coding viral RNAs, such as the 3¢ non- coding region of a viral genome used for transformation (Zaccomer et al., 1993), have also been shown to confer resistance through competition with the viral RNAs.

However, the most important type of RNA-mediated resistance is produced via a quite different mechanism, fi rst denoted as gene silencing, now most often termed RNA interference, or RNAi. This was fi rst shown by Dougherty’s group, who demonstrated that resistance to Tobacco etch virus could be conferred by an untranslatable CP gene, and also that the strongest RNA-mediated protection was observed in plant lines in which little or no transgene mRNA accu- mulated. When resistance was observed in plants that initially accumulated transgene mRNA, the plants were fi rst fully infected, after which, recovery, leading to a com- pletely insensitive state, was accompanied by disappearance of the transgene mRNA. They proposed that resistance was con- ferred by sequence-specifi c degradation of both the transgene mRNA and the corre-


 

sponding viral RNA, via a mechanism sim- ilar to post-transcriptional gene silencing (PTGS) (Lindbo et al., 1993; Dougherty et al., 1994).

Subsequently, gene silencing, or RNAi, has been the subject of many investigations. Basically, gene silencing is thought to result from the action of a mechanism that sur- veys the RNAs in a cell and sequence specifi cally degrades those perceived as unwanted. This phenomenon has been pro- posed to be a natural plant defence mecha- nism against viruses and transposons and is also involved in the regulation of the expression levels of certain genes (Waterhouse et al., 2001; Tang et al., 2002; Voinnet, 2002). Evidence of this is that some plant viruses encode proteins that specifi cally disable this system.

RNA silencing (under different names) has been demonstrated in animals, fungi, plants (Fig. 14.2) and possibly in bacteria (Tchurikov et al., 2000). The mechanism involved in these organisms shares some common features, but the hallmark is the production of small (21–25 nucleotide) RNAs that act as determinants for the RNA degradation (Hamilton and Baulcombe, 1999; Hammond et al., 2000). In plant cells infected by a virus, the RNA fi rst requires the conversion to double-stranded (ds) RNA. This is probably accomplished by the viral replicase, although transgenes with inverted repeats may directly produce dsRNA. The dsRNA is cleaved into the small interfering RNAs (siRNAs) that are the mediators of gene silencing by an RNase III-like enzyme complex (Dicer) that is ATP dependent and contains putative helicase-, RNase III- and dsRNA-binding domains (Fagard and Vaucheret, 2000; Park et al., 2002). Two classes of siRNA are generated in plants by different Dicer enzymes, short siRNA (21 nucleotides) and long siRNA (24 nucleotides) (Hamilton and Baulcombe, 1999; Tang et al., 2002). These siRNAs are double stranded with two- nucleotide 3¢ overhangs and hydroxyl ter- mini. It has been proposed that the short siRNAs direct PTGS via mRNA degradation and the long siRNAs trigger systemic


 

 

Fig. 14.2. A model for RNA silencing in transgenic plants. An inverted repeat copy of a gene with homology to the target viral sequence forms a double-stranded RNA (dsRNA) molecule upon expression. In the case of single copy transgenes, the mRNA can be perceived as aberrant by an unknown mechanism and is converted to dsRNA by a plant-specifi c RNA-dependent RNA polymerase (RdRp). The dsRNA is translocated to the cytoplasm where it becomes a target for the Dicer enzyme. Viral replication of many RNA viruses also includes a dsRNA replicative form that can be targeted by Dicer. In plants, two types of Dicer proteins generate short (21 nucleotide) and long (25 nucleotide) micro RNAs (miRNA, also denominated small interfering RNA, siRNA). These siRNAs direct RNA interference in plants. The short ones are associated with local silencing and the long ones with systemic silencing. The short siRNAs associate with a RISC protein and become single stranded in an activation process that requires ATP. The RISC–siRNA complex directs the sequence-specifi c degradation of homologous RNA. In the diagram, the viral RNA is degraded by the transgene-induced RNA silencing (dotted arrow). It should be noted that the process of degradation can also occur in the nucleus.

 

 


silencing and the methylation of homolo- gous DNA (Hamilton et al., 2002). The siRNAs are opened in an ATP-dependent step, leaving them single stranded to be incorporated into the multimeric RNase complex, that is then denominated RISC (RNA interference-specific complex), which is guided by Watson–Crick base paring, ensuring the sequence-specific degradation of the unwanted RNA (Baulcombe, 2002; Wassenegger, 2002). Inducible endogenous plant RNA-depend- ent RNA polymerases (RdRps) are also involved in RNA silencing. In both


Arabidopsis and tobacco, virus- and SA- induced RdRps have been identifi ed that are not required for the initiation of RNA silencing but are necessary for its mainte- nance (Yu et al., 2003).

In plants, constructs designed to pro- duce dsRNA or self-complementary hairpin RNA transcribed from inverted repeats have been shown to be highly effi cient inducers of gene silencing. This same strat- egy can lead to very effective virus resist- ance or immunity (Waterhouse et al., 1998; Smith et al., 2000; Wang et al., 2000; Tenllado et al., 2004).


 


 






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