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Viroid Pathogenesis
C H A P T E R
9 Viroid Pathogenesis Ricardo Flores1, Francesco Di Serio2, Beatriz Navarro2 and Robert A. Owens3 1
Polytechnic University of Valencia-CSIC, Valencia, Spain 2 National Research Council, Bari, Italy 3 U.S. Department of Agriculture, Beltsville, MD, United States
INTRODUCTION From early research on viroids, “How do viroids make plants sick?” has been a question of interest. Most symptoms associated with viroid diseases can also be seen in plants infected with viruses, and the small size and noncoding nature of the viroid genome requires that viroidinduced disease be an essentially host-generated process. Previous reviews by three viroid pioneers (e.g., Diener, 1987; Sa¨nger, 1982; Semancik, 2003) summarized information from the early, observational phases of work on viroid pathogenicity. Soon after the first complete viroid sequence was reported (Gross et al., 1978), speculation describing possible molecular mechanism(s) of disease induction appeared. In the absence of viroid-encoded polypeptides, disease was assumed to result from the direct interaction of the viroid (or its complement) with cellular constituents. Only later were indirect interactions between viroid and host via newly discovered regulatory pathways like RNA silencing considered. Initially, very little experimental data was available to test these hypotheses. More recently, the situation has changed, and in this chapter we summarize evidence indicating the involvement of multiple molecular mechanisms in viroid disease induction. Promising strategies to expand our understanding of the linkages among different aspects of the host response are also described.
Viroids and Satellites. DOI: http://dx.doi.org/10.1016/B978-0-12-801498-1.00009-7
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MACROSCOPIC SYMPTOMS AND CYTOPATHIC EFFECTS Although many viroids were discovered due to their ability to cause disease, not all viroid infections are symptomatic. Stunting is often considered a characteristic of viroid disease, but additional symptoms may include epinasty, distortions affecting leaves and flowers, veinal necrosis, bark disorders, changes in fruit shape/color, sterility, and rapid aging (see Chapter 5: Viroid Biology). The root systems of infected plants are often reduced in size and reserve organs, like tubers, may also be misshapen. For many viroids, symptom expression can be enhanced by high temperatures—in contrast to most plant viruses. Only in coconut cadang-cadang and Tinangaja diseases does infection result in death of the whole plant. At the microscopic level, reported cytopathic effects of viroid infection include: (1) proliferation of cytoplasmic membranes to form “plasmalemmasomes”; (2) distortion of cell walls; (3) chloroplast abnormalities associated with members of both families; and (4) formation of electron-dense deposits (in the cytoplasm and chloroplasts) (Di Serio et al., 2013). How these effects are related to symptoms at the whole plant level remains unclear, but disruption of chloroplast function is also a prominent feature of many plant virus infections (see Chapter 10: Changes in the Host Proteome and Transcriptome Induced by Viroid Infection).
BIOCHEMICAL CHANGES ACCOMPANYING VIROID INFECTION Many symptoms associated with viroid infection (e.g., stunting) are consistent with disturbances in hormone metabolism. Evidence of hormonal changes has been presented for tomato infected with citrus exocortis viroid (CEVd) (Duran-Vila and Semancik, 1982) and cucumber infected with hop stunt viroid (HSVd) (Yaguchi and Takahashi, 1985). Other changes in the host metabolome include (1) increased levels of gentisic acid (a signaling molecule related to salicylic acid) in CEVdinfected tomato (Belle´s et al., 1999) and (2) reduced alpha acid content of hop cones harvested from HSVd-infected vines (see Chapter 19: Hop Stunt Viroid). Other alterations affect host macromolecules. Among the first reported were changes in two low molecular weight pathogenesisrelated (PR) proteins in CEVd-infected Gynura aurantiaca (Conejero and Semancik, 1977). PR protein synthesis is one component of systemic
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acquired resistance, a term describing a coordinated series of plant responses to pathogen attack. Additional information concerning PR protein accumulation in viroid-infected plants can be found in Chapter 10, Changes in the Host Proteome and Transcriptome Induced by Viroid Infection. Other proteins are modified (e.g., phosphorylated; Hiddinga et al., 1988) in response to viroid infection. Overexpression in tobacco of a signal-transducing tomato protein kinase (PKV), resulted in stunting, modified vascular development, reduced root formation, and male sterility (Hammond and Zhao, 2009). PKV may regulate plant development by modulating critical signaling pathways involved in gibberellic acid metabolism.
MOLECULAR MECHANISMS Determination of the complete nucleotide sequence of potato spindle tuber viroid (PSTVd) (Gross et al., 1978) was a landmark in plant biology. Certain similarities with U1 small nuclear RNA and eukaryotic introns suggested a possible mechanism for pathogenicity (Diener, 1981). Two further developments—the finding that minor sequence differences distinguish PSTVd strains inducing very different symptoms (Gross et al., 1981) and construction of infectious PSTVd-cDNA clones (Cress et al., 1983)—caused the emphasis in pathogenicity research to shift, turning away from the host to focus almost exclusively on the viroid. Site-directed mutagenesis allowed experimental testing of mechanisms proposed for viroid pathogenesis. As sequences of other members of the family Pospiviroidae became available, Keese and Symons (1985) proposed that their rod-like genomes contain five structural/functional domains, one of which is the pathogenicity (P) domain. For some viroids, sequences outside the P domain also play an important role in symptom expression (e.g., Sano et al., 1992). Even for PSTVd the situation has proved to be more complex than originally believed; thus, a single nucleotide substitution in the loop E motif of the central domain can either (1) broaden the host range to include tobacco (Wassenegger et al., 1996) or (2) modify the tomato symptoms to produce a “flat top” phenotype (Qi and Ding, 2003). Schno¨lzer et al. (1985) proposed that a “virulence-modulating” region within the P domain of PSTVd modulates symptoms by interacting with unidentified host constituents. Diener et al. (1993) described a differential activation of the interferon-induced, dsRNA-activated, mammalian protein kinase by PSTVd strains of varying pathogenicity, but efforts to clone a plant homolog have been unsuccessful.
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THE HOST CONTRIBUTION TO PATHOGENICITY REEXAMINED Skoric et al. (2001), using two stable, naturally occurring sequence variants of CEVd, observed that increasing the daytime temperature from 35 to 40 C caused symptoms of the mild (but not the severe) variant to intensify; furthermore, this effect was (1) reversible and (2) only observed when the host used was G. aurantiaca. Structural calculations failed to reveal any linkage with predicted CEVd secondary structures. Modern genomic techniques such as microarray or proteomic analysis have been used to examine changes in host gene expression associated with viroid infection (see Chapter 10: Changes in the Host Proteome and Transcriptome Induced by Viroid Infection). In tomato (where analysis is facilitated by the availability of a complete genomic sequence), PSTVd induces numerous changes in mRNAs encoding enzymes involved in hormone biosynthesis and components of the corresponding signaling pathways (Owens et al., 2012). Using a combination of proteomic analysis and qRT-PCR, Liso´n et al. (2013) have shown that control of the accumulation of some proteins resides at the translational rather than transcriptional level.
RNA SILENCING LIMITS THE SPREAD OF VIROID INFECTION Discovery of RNA silencing, first in plants and subsequently in most other eukaryotes, revealed novel regulatory roles for RNA (apart from those involved in protein translation) and integrated diverse observations into a single interpretative scheme. This topic is discussed extensively in Chapter 11, Viroids and RNA Silencing. In brief, RNA silencing most likely emerged as a defense mechanism against invading agents and then was coopted for other roles including regulation of gene expression. Transcriptional and posttranscriptional gene silencing are initiated by dsRNA, which in plants are cleaved by Dicer-like (DCL) enzymes into small RNAs (sRNAs): microRNAs (miRNAs, 2122 nt) of endogenous origin and small interfering RNAs (siRNAs, 21, 22 or 24 nt) of both endogenous and exogenous origin (Axtell, 2013). One strand of the sRNA duplex guides the RNA inducing silencing complex (RISC), to inactivate either complementary RNA or DNA. Plant RNA and DNA viruses are restrained by the combined action of DCLs and RISC. RNA silencing also restrains viroid infection, as shown by the detection (Itaya et al., 2001; Martı´nez de Alba et al., 2002; Papaefthimiou et al., 2001) and characterization (Di Serio et al., 2009; Itaya et al., 2007;
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Machida et al., 2007; Martı´n et al., 2007; Navarro et al., 2009) of viroidderived sRNAs (vd-sRNAs) in tissues infected by members of both viroid families. These vd-sRNAs reduce, in a sequence-specific manner, the expression of a reporter gene (Itaya et al., 2007; Vogt et al., 2004), and the viroid titer in infected plants (Carbonell et al., 2008). Moreover, one specific RNA-directed RNA polymerase (RDR6) acts to reduce the titer of PSTVd and block its entry into meristems (Di Serio et al., 2010). These data, particularly the sequence-specific effects of vd-sRNAs, suggest that (1) a “dicing-only” mechanism is unlikely and (2) one or more Argonaute proteins (AGOs) (Mallory and Vaucheret, 2010), at the core of RISC, are also involved. Indeed, pioneering work revealed that PSTVd-cDNA sequences in transgenic tobacco become methylated de novo in an RNA-directed sequence-specific manner following infection and replication of PSTVd (Wassenegger et al., 1994), thus providing the first example of transcriptional gene silencing, subsequently shown to be mediated by specific DCL-generated siRNAs and AGOs. There is direct evidence that AGOs recruit vd-sRNAs (Minoia et al., 2014). In PSTVd-infected Nicotiana benthamiana, both endogenous AGO1 and epitope-tagged Arabidopsis thaliana AGO1, AGO2, AGO4, and AGO5 associate with vd-sRNAs displaying the same properties (50 -terminal nucleotide and size) as endogenous and viral sRNAs (Mi et al., 2008). Furthermore, overexpression of these four AGOs attenuated viroid accumulation, supporting the involvement of RISC in antiviroid defense and consistent with other in vitro results showing that RISC mediates cleavage of viral RNAs with a compact conformation—like those of viroids—by targeting bulged regions within this conformation (Schuck et al., 2013).
RNA SILENCING ALSO MEDIATES VIROID PATHOGENESIS In addition to guiding RISC against viroid RNAs, vd-sRNAs could also target specific host mRNAs, whose inactivation would ultimately result in symptom development through a signal transduction cascade (Go´mez et al., 2009; Papaefthimiou et al., 2001; Wang et al., 2004). This intriguing hypothesis has gathered increasing experimental support. For viroids that replicate in plastids (family Avsunviroidae), certain variants of peach latent mosaic viroid (PLMVd) containing a specific 1214 nt insertion elicit an extreme albinism known as “peach calico” (PC) (Chapter 29: Peach Latent Mosaic Viroid in Infected Peach; Malfitano et al., 2003; Rodio et al., 2007). This insertion contains the determinant for PC, and the initial defect is most likely triggered by two 21-nt PLMVd-sRNAs that include the pathogenic determinant and
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differ in one position. The two PLMVd-sRNAs are complementary to a 21-nt sequence in the peach mRNA coding for the chloroplastic heatshock protein 90 (cHSP90), and target it for cleavage at the expected site (between positions 10 and 11 of the PLMVd-sRNAs). cHSP90 is a chaperone that mediates chloroplast biogenesis (a process impaired in PC-affected tissue); furthermore, a single-amino acid mutation in cHSP90 induces a yellow phenotype in A. thaliana. Hence, these results provide a plausible mechanism for the initiation of PC pathogenesis (Navarro et al., 2012). An account of the properties of the two vd-sRNA that support their involvement in RNA silencingmediated cleavage of cHSP90 mRNA has been reported elsewhere (Flores et al., 2015). Prominent among these properties is the presence of a 50 -terminal U, indicating that these viroid sRNAs most likely bind and guide AGO1 (Mi et al., 2008), which plays a key role in RNA silencing via cleavage or translation arrest of endogenous and invading RNA targets (Baumberger and Baulcombe, 2005; Morel et al., 2002). For viroids that replicate in the nucleus (family Pospiviroidae), a similar RNA silencing mechanism has been proposed for PSTVd and tomato planta macho viroid (TPMVd). For PSTVd, constitutive expression of artificial miRNAs derived from one of its pathogenicity determinants induces abnormal phenotypes in two Nicotiana species (Eamens et al., 2014), and vd-sRNAs from this determinant silence callose synthase genes in tomato (Adkar-Purushothama et al., 2015). Results for TPMVd are based on in silico prediction and experimental validation of the targeting of a tomato mRNA by vd-sRNA from the same determinant (Avina-Padilla et al., 2015). How inactivation of the mRNAs targeted— coding for a soluble inorganic pyrophosphatase, callose synthases, and a WD40-repeat protein, respectively—leads to disease induction is not yet clear. Also unclear is the AGO involved, considering that most of the vd-sRNAs lack a 50 -terminal U. Finally, infection of cucumber by HSVd results in dynamic changes in the methylation status of ribosomal RNA promotor sequences (Martı´nez et al., 2014). The potential role of these changes in pathogenesis remains to be examined.
VIROID PATHOGENESIS ENGAGES MULTIPLE REGULATORY NETWORKS The biochemical and molecular mechanisms used by plants to defend themselves against attack by diverse pathogens (i.e., fungi, bacteria, viruses, and viroids) exhibit many common features (Hammond-Kosack and Jones, 2015). For example, photosynthetic activity almost always decreases following infection, probably as part of a host effort to conserve energy resources needed to mount the defense response. The
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studies described above have identified single host genes that respond to signals originating from the viroid genome, either directly (via interaction with host proteins) or indirectly (via RNA silencing). How the effects of viroid infection spread from the limited number of initial interactions characterized thus far to affect diverse metabolic pathways is currently unknown. As discussed in Chapter 10, Changes in the Host Proteome and Transcriptome Induced by Viroid Infection, microarray analysis of PSTVd-infected “Rutgers” tomato has revealed that infection-related changes in transcript levels involve more than half of the approximately 10,000 genes present on the array. Among the genes affected were those encoding enzymes involved in biosynthesis of gibberellin and other hormones as well as components of their respective signaling pathways. Proteomic analysis of CEVd-infected tomato has shown that posttranscriptional changes may involve translational arrest/inhibition as well as sRNA-mediated mRNA degradation. Large-scale sRNA sequence analyses have also revealed changes in host miRNA levels following viroid infection. Direct evidence for cleavage of the target genes is limited; nevertheless, the list of potential target genes includes a number of transcription factors and other regulatory proteins likely to occupy hub positions in the still-to-be-determined host interactome. The picture of viroid pathogenicity that has begun to emerge is clearly complex, with regulatory controls operating at multiple levels. To date, genome-wide studies involving viroids have focused on the host transcriptome. However, integration of transcriptional and proteomic data sets may yield novel perspectives (Payne, 2015). Until recently, transcriptional and proteinprotein interaction networks were available only for A. thaliana, but similar attempts with other plant species are expanding rapidly (Braun et al., 2013). A recent meta-analysis comparing the effects of eight plant viruses on A. thaliana gene expression (Rodrigo et al., 2012) illustrates the benefits of such an approach and is discussed in more detail in Chapter 10, Changes in the Host Proteome and Transcriptome Induced by Viroid Infection. The data sets and tools required to compare the effects of virus and viroid infections in tomato should soon become available, and the expected results should provide a much more detailed picture of viroid pathogenicity.
Acknowledgments Research in R.F. laboratory is currently funded by grant BFU201456812-P from the Spanish Ministerio de Economı´a y Competitividad (MINECO). Research in F.D.S. and B.N laboratory has been partially supported by a dedicated grant (CISIA) of the Ministero dell’Economia e Finanze Italiano to the CNR (Legge n. 191/2009).
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9 Viroid Pathogenesis Ricardo Flores1, Francesco Di Serio2, Beatriz Navarro2 and Robert A. Owens3 1
Polytechnic University of Valencia-CSIC, Valencia, Spain 2 National Research Council, Bari, Italy 3 U.S. Department of Agriculture, Beltsville, MD, United States
INTRODUCTION From early research on viroids, “How do viroids make plants sick?” has been a question of interest. Most symptoms associated with viroid diseases can also be seen in plants infected with viruses, and the small size and noncoding nature of the viroid genome requires that viroidinduced disease be an essentially host-generated process. Previous reviews by three viroid pioneers (e.g., Diener, 1987; Sa¨nger, 1982; Semancik, 2003) summarized information from the early, observational phases of work on viroid pathogenicity. Soon after the first complete viroid sequence was reported (Gross et al., 1978), speculation describing possible molecular mechanism(s) of disease induction appeared. In the absence of viroid-encoded polypeptides, disease was assumed to result from the direct interaction of the viroid (or its complement) with cellular constituents. Only later were indirect interactions between viroid and host via newly discovered regulatory pathways like RNA silencing considered. Initially, very little experimental data was available to test these hypotheses. More recently, the situation has changed, and in this chapter we summarize evidence indicating the involvement of multiple molecular mechanisms in viroid disease induction. Promising strategies to expand our understanding of the linkages among different aspects of the host response are also described.
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MACROSCOPIC SYMPTOMS AND CYTOPATHIC EFFECTS Although many viroids were discovered due to their ability to cause disease, not all viroid infections are symptomatic. Stunting is often considered a characteristic of viroid disease, but additional symptoms may include epinasty, distortions affecting leaves and flowers, veinal necrosis, bark disorders, changes in fruit shape/color, sterility, and rapid aging (see Chapter 5: Viroid Biology). The root systems of infected plants are often reduced in size and reserve organs, like tubers, may also be misshapen. For many viroids, symptom expression can be enhanced by high temperatures—in contrast to most plant viruses. Only in coconut cadang-cadang and Tinangaja diseases does infection result in death of the whole plant. At the microscopic level, reported cytopathic effects of viroid infection include: (1) proliferation of cytoplasmic membranes to form “plasmalemmasomes”; (2) distortion of cell walls; (3) chloroplast abnormalities associated with members of both families; and (4) formation of electron-dense deposits (in the cytoplasm and chloroplasts) (Di Serio et al., 2013). How these effects are related to symptoms at the whole plant level remains unclear, but disruption of chloroplast function is also a prominent feature of many plant virus infections (see Chapter 10: Changes in the Host Proteome and Transcriptome Induced by Viroid Infection).
BIOCHEMICAL CHANGES ACCOMPANYING VIROID INFECTION Many symptoms associated with viroid infection (e.g., stunting) are consistent with disturbances in hormone metabolism. Evidence of hormonal changes has been presented for tomato infected with citrus exocortis viroid (CEVd) (Duran-Vila and Semancik, 1982) and cucumber infected with hop stunt viroid (HSVd) (Yaguchi and Takahashi, 1985). Other changes in the host metabolome include (1) increased levels of gentisic acid (a signaling molecule related to salicylic acid) in CEVdinfected tomato (Belle´s et al., 1999) and (2) reduced alpha acid content of hop cones harvested from HSVd-infected vines (see Chapter 19: Hop Stunt Viroid). Other alterations affect host macromolecules. Among the first reported were changes in two low molecular weight pathogenesisrelated (PR) proteins in CEVd-infected Gynura aurantiaca (Conejero and Semancik, 1977). PR protein synthesis is one component of systemic
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acquired resistance, a term describing a coordinated series of plant responses to pathogen attack. Additional information concerning PR protein accumulation in viroid-infected plants can be found in Chapter 10, Changes in the Host Proteome and Transcriptome Induced by Viroid Infection. Other proteins are modified (e.g., phosphorylated; Hiddinga et al., 1988) in response to viroid infection. Overexpression in tobacco of a signal-transducing tomato protein kinase (PKV), resulted in stunting, modified vascular development, reduced root formation, and male sterility (Hammond and Zhao, 2009). PKV may regulate plant development by modulating critical signaling pathways involved in gibberellic acid metabolism.
MOLECULAR MECHANISMS Determination of the complete nucleotide sequence of potato spindle tuber viroid (PSTVd) (Gross et al., 1978) was a landmark in plant biology. Certain similarities with U1 small nuclear RNA and eukaryotic introns suggested a possible mechanism for pathogenicity (Diener, 1981). Two further developments—the finding that minor sequence differences distinguish PSTVd strains inducing very different symptoms (Gross et al., 1981) and construction of infectious PSTVd-cDNA clones (Cress et al., 1983)—caused the emphasis in pathogenicity research to shift, turning away from the host to focus almost exclusively on the viroid. Site-directed mutagenesis allowed experimental testing of mechanisms proposed for viroid pathogenesis. As sequences of other members of the family Pospiviroidae became available, Keese and Symons (1985) proposed that their rod-like genomes contain five structural/functional domains, one of which is the pathogenicity (P) domain. For some viroids, sequences outside the P domain also play an important role in symptom expression (e.g., Sano et al., 1992). Even for PSTVd the situation has proved to be more complex than originally believed; thus, a single nucleotide substitution in the loop E motif of the central domain can either (1) broaden the host range to include tobacco (Wassenegger et al., 1996) or (2) modify the tomato symptoms to produce a “flat top” phenotype (Qi and Ding, 2003). Schno¨lzer et al. (1985) proposed that a “virulence-modulating” region within the P domain of PSTVd modulates symptoms by interacting with unidentified host constituents. Diener et al. (1993) described a differential activation of the interferon-induced, dsRNA-activated, mammalian protein kinase by PSTVd strains of varying pathogenicity, but efforts to clone a plant homolog have been unsuccessful.
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THE HOST CONTRIBUTION TO PATHOGENICITY REEXAMINED Skoric et al. (2001), using two stable, naturally occurring sequence variants of CEVd, observed that increasing the daytime temperature from 35 to 40 C caused symptoms of the mild (but not the severe) variant to intensify; furthermore, this effect was (1) reversible and (2) only observed when the host used was G. aurantiaca. Structural calculations failed to reveal any linkage with predicted CEVd secondary structures. Modern genomic techniques such as microarray or proteomic analysis have been used to examine changes in host gene expression associated with viroid infection (see Chapter 10: Changes in the Host Proteome and Transcriptome Induced by Viroid Infection). In tomato (where analysis is facilitated by the availability of a complete genomic sequence), PSTVd induces numerous changes in mRNAs encoding enzymes involved in hormone biosynthesis and components of the corresponding signaling pathways (Owens et al., 2012). Using a combination of proteomic analysis and qRT-PCR, Liso´n et al. (2013) have shown that control of the accumulation of some proteins resides at the translational rather than transcriptional level.
RNA SILENCING LIMITS THE SPREAD OF VIROID INFECTION Discovery of RNA silencing, first in plants and subsequently in most other eukaryotes, revealed novel regulatory roles for RNA (apart from those involved in protein translation) and integrated diverse observations into a single interpretative scheme. This topic is discussed extensively in Chapter 11, Viroids and RNA Silencing. In brief, RNA silencing most likely emerged as a defense mechanism against invading agents and then was coopted for other roles including regulation of gene expression. Transcriptional and posttranscriptional gene silencing are initiated by dsRNA, which in plants are cleaved by Dicer-like (DCL) enzymes into small RNAs (sRNAs): microRNAs (miRNAs, 2122 nt) of endogenous origin and small interfering RNAs (siRNAs, 21, 22 or 24 nt) of both endogenous and exogenous origin (Axtell, 2013). One strand of the sRNA duplex guides the RNA inducing silencing complex (RISC), to inactivate either complementary RNA or DNA. Plant RNA and DNA viruses are restrained by the combined action of DCLs and RISC. RNA silencing also restrains viroid infection, as shown by the detection (Itaya et al., 2001; Martı´nez de Alba et al., 2002; Papaefthimiou et al., 2001) and characterization (Di Serio et al., 2009; Itaya et al., 2007;
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Machida et al., 2007; Martı´n et al., 2007; Navarro et al., 2009) of viroidderived sRNAs (vd-sRNAs) in tissues infected by members of both viroid families. These vd-sRNAs reduce, in a sequence-specific manner, the expression of a reporter gene (Itaya et al., 2007; Vogt et al., 2004), and the viroid titer in infected plants (Carbonell et al., 2008). Moreover, one specific RNA-directed RNA polymerase (RDR6) acts to reduce the titer of PSTVd and block its entry into meristems (Di Serio et al., 2010). These data, particularly the sequence-specific effects of vd-sRNAs, suggest that (1) a “dicing-only” mechanism is unlikely and (2) one or more Argonaute proteins (AGOs) (Mallory and Vaucheret, 2010), at the core of RISC, are also involved. Indeed, pioneering work revealed that PSTVd-cDNA sequences in transgenic tobacco become methylated de novo in an RNA-directed sequence-specific manner following infection and replication of PSTVd (Wassenegger et al., 1994), thus providing the first example of transcriptional gene silencing, subsequently shown to be mediated by specific DCL-generated siRNAs and AGOs. There is direct evidence that AGOs recruit vd-sRNAs (Minoia et al., 2014). In PSTVd-infected Nicotiana benthamiana, both endogenous AGO1 and epitope-tagged Arabidopsis thaliana AGO1, AGO2, AGO4, and AGO5 associate with vd-sRNAs displaying the same properties (50 -terminal nucleotide and size) as endogenous and viral sRNAs (Mi et al., 2008). Furthermore, overexpression of these four AGOs attenuated viroid accumulation, supporting the involvement of RISC in antiviroid defense and consistent with other in vitro results showing that RISC mediates cleavage of viral RNAs with a compact conformation—like those of viroids—by targeting bulged regions within this conformation (Schuck et al., 2013).
RNA SILENCING ALSO MEDIATES VIROID PATHOGENESIS In addition to guiding RISC against viroid RNAs, vd-sRNAs could also target specific host mRNAs, whose inactivation would ultimately result in symptom development through a signal transduction cascade (Go´mez et al., 2009; Papaefthimiou et al., 2001; Wang et al., 2004). This intriguing hypothesis has gathered increasing experimental support. For viroids that replicate in plastids (family Avsunviroidae), certain variants of peach latent mosaic viroid (PLMVd) containing a specific 1214 nt insertion elicit an extreme albinism known as “peach calico” (PC) (Chapter 29: Peach Latent Mosaic Viroid in Infected Peach; Malfitano et al., 2003; Rodio et al., 2007). This insertion contains the determinant for PC, and the initial defect is most likely triggered by two 21-nt PLMVd-sRNAs that include the pathogenic determinant and
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differ in one position. The two PLMVd-sRNAs are complementary to a 21-nt sequence in the peach mRNA coding for the chloroplastic heatshock protein 90 (cHSP90), and target it for cleavage at the expected site (between positions 10 and 11 of the PLMVd-sRNAs). cHSP90 is a chaperone that mediates chloroplast biogenesis (a process impaired in PC-affected tissue); furthermore, a single-amino acid mutation in cHSP90 induces a yellow phenotype in A. thaliana. Hence, these results provide a plausible mechanism for the initiation of PC pathogenesis (Navarro et al., 2012). An account of the properties of the two vd-sRNA that support their involvement in RNA silencingmediated cleavage of cHSP90 mRNA has been reported elsewhere (Flores et al., 2015). Prominent among these properties is the presence of a 50 -terminal U, indicating that these viroid sRNAs most likely bind and guide AGO1 (Mi et al., 2008), which plays a key role in RNA silencing via cleavage or translation arrest of endogenous and invading RNA targets (Baumberger and Baulcombe, 2005; Morel et al., 2002). For viroids that replicate in the nucleus (family Pospiviroidae), a similar RNA silencing mechanism has been proposed for PSTVd and tomato planta macho viroid (TPMVd). For PSTVd, constitutive expression of artificial miRNAs derived from one of its pathogenicity determinants induces abnormal phenotypes in two Nicotiana species (Eamens et al., 2014), and vd-sRNAs from this determinant silence callose synthase genes in tomato (Adkar-Purushothama et al., 2015). Results for TPMVd are based on in silico prediction and experimental validation of the targeting of a tomato mRNA by vd-sRNA from the same determinant (Avina-Padilla et al., 2015). How inactivation of the mRNAs targeted— coding for a soluble inorganic pyrophosphatase, callose synthases, and a WD40-repeat protein, respectively—leads to disease induction is not yet clear. Also unclear is the AGO involved, considering that most of the vd-sRNAs lack a 50 -terminal U. Finally, infection of cucumber by HSVd results in dynamic changes in the methylation status of ribosomal RNA promotor sequences (Martı´nez et al., 2014). The potential role of these changes in pathogenesis remains to be examined.
VIROID PATHOGENESIS ENGAGES MULTIPLE REGULATORY NETWORKS The biochemical and molecular mechanisms used by plants to defend themselves against attack by diverse pathogens (i.e., fungi, bacteria, viruses, and viroids) exhibit many common features (Hammond-Kosack and Jones, 2015). For example, photosynthetic activity almost always decreases following infection, probably as part of a host effort to conserve energy resources needed to mount the defense response. The
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studies described above have identified single host genes that respond to signals originating from the viroid genome, either directly (via interaction with host proteins) or indirectly (via RNA silencing). How the effects of viroid infection spread from the limited number of initial interactions characterized thus far to affect diverse metabolic pathways is currently unknown. As discussed in Chapter 10, Changes in the Host Proteome and Transcriptome Induced by Viroid Infection, microarray analysis of PSTVd-infected “Rutgers” tomato has revealed that infection-related changes in transcript levels involve more than half of the approximately 10,000 genes present on the array. Among the genes affected were those encoding enzymes involved in biosynthesis of gibberellin and other hormones as well as components of their respective signaling pathways. Proteomic analysis of CEVd-infected tomato has shown that posttranscriptional changes may involve translational arrest/inhibition as well as sRNA-mediated mRNA degradation. Large-scale sRNA sequence analyses have also revealed changes in host miRNA levels following viroid infection. Direct evidence for cleavage of the target genes is limited; nevertheless, the list of potential target genes includes a number of transcription factors and other regulatory proteins likely to occupy hub positions in the still-to-be-determined host interactome. The picture of viroid pathogenicity that has begun to emerge is clearly complex, with regulatory controls operating at multiple levels. To date, genome-wide studies involving viroids have focused on the host transcriptome. However, integration of transcriptional and proteomic data sets may yield novel perspectives (Payne, 2015). Until recently, transcriptional and proteinprotein interaction networks were available only for A. thaliana, but similar attempts with other plant species are expanding rapidly (Braun et al., 2013). A recent meta-analysis comparing the effects of eight plant viruses on A. thaliana gene expression (Rodrigo et al., 2012) illustrates the benefits of such an approach and is discussed in more detail in Chapter 10, Changes in the Host Proteome and Transcriptome Induced by Viroid Infection. The data sets and tools required to compare the effects of virus and viroid infections in tomato should soon become available, and the expected results should provide a much more detailed picture of viroid pathogenicity.
Acknowledgments Research in R.F. laboratory is currently funded by grant BFU201456812-P from the Spanish Ministerio de Economı´a y Competitividad (MINECO). Research in F.D.S. and B.N laboratory has been partially supported by a dedicated grant (CISIA) of the Ministero dell’Economia e Finanze Italiano to the CNR (Legge n. 191/2009).
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