GENETICS OF VIRUS RESISTANCE IN NATURE

The first step in the study of genetics of viral resistance is to determine whether the resistant response is inherited, and if so, the number of genes involved and their mode of inheritance. [For reviews on sources of host resistance to plant viruses and inheritance of resistance to plant viruses and viral disease, for underlying general trends or common mechanisms of virus resistance, and for specific crop or viral groups. Over 200 virus R genes reported in studies of crops, their wild relatives, and the model species Arabidopsis thaliana, as well as both inheritance and information about possible mechanisms, where known, are included. Genes reported to show dominant inheritance are listed in Supplementary Table 1, genes reported to show recessive inheritance are listed in Supplementary Table 2. This discussion highlights key observations drawn primarily from studies of dicot species, the focus of most work to date. Within the dicots, information regarding several plant families, notably the Solanaceae, Cucurbitaceae, and Leguminosae, predominate for historical and agricultural reasons. R genes reported from monocot species are almost exclusively limited to major crop species, e.g., barley, wheat, and rice. More than 80% of reported viral resistance is monogenically controlled; the remainder shows oligogenic or polygenic control. Only slightly more than half of all reported monogenic resistance traits show dominant inheritance. In most but not all cases, dominance has

Table 1.Some cross generic R gene groups that include virus resistance genes in Solanaceae - Chromosomes and resistance genes

Plant	Chr 4	Chr 5	Chr 7	Chr 10	Chr 11	Chr 12
Tomato	Hero (n)		Ph-1 (p)	Ph-2 (p)	Ty-2 (v), I2 (f),    Sm (f)
Potato	R2 (p), Ny (v)	Grp1 (n), Gpa (n), Gpa5 (n),    R1 (p),    phyt3 (p), Pi1(p), Rx2 (v), Nb (v)	Gro1 (n), Rpi1, R9 (p)	Gro1.2 (n)	R3 (p), R6 (p), R7 (p), Gro1.3(n),     phyt7 (p)	Gpa2 (n), Rx1 (v)
Pepper	Phyto5.2 (p)				L (v), cmv11.1(v), phyt3 (p)

Positions are inferred, so the precise linkage relationships of these loci remain unclear in many cases. Gene names are followed by letters designating the major pathogen group controlled:(b) bacteria, (f) fungi, (n) nematode, (p) Phytophthora spp., (v) virus.(Kang B-C et al. 2005. Annu Rev Phytopathol 43: 581-62).

been reported as complete. The heterozygote may show a clearly different response from that of the homozygote, however this is rarely checked carefully in inheritance studies. Where incomplete dominance is observed, there are important implications for mechanisms that may involve gene dosage effects. The relatively high proportion of recessive viral R genes is in marked contrast to fungal or bacterial resistance where most reported resistance is dominant. About one third of the R genes listed in Supplementary Tables 1 and 2, have been tagged with molecular genetic markers including RFLP, AFLP, RAPD, and various other PCR-based markers. Molecular markers linked to R genes can be used for indirect selection via genotype, for locating R genes in plant genomes and for gene isolation. Relatively few quantitative trait loci (QTL) for plant viral resistance have been tagged or genetically mapped. Both pathogen and host taxa are composed of dynamic populations and therefore unambiguous identification of host and pathogen genotypes is essential, ideally with representative genotypes archived in a stable and reliable location such as the USDA National Plant Germplasm System or the American Type Culture Collection. Historically, important collections of plant germplasm and viral cultures have

been maintained at universities and research institutes, where shifts in staffing and resource allocation may put critical genetic resources at risk. Within a host gene pool, there may be several to many independent sources of resistance to a single virus or viral pathotype (a set of viral genotypes that interact similarly with a set of host lines showing differential response).

 The advent of molecular methods has demonstrated that these R genes may represent different loci with shared or independent evolutionary histories, or different alleles at the same locus. Numerous early studies concluded that R genes with different resistance specificities necessarily occurred at distinct genetic loci; however, this is clearly not the case. Whenever there is overlap

Table 2.Examples of host factors involved in plant viral life cycles and resistance mechanisms

Movement type	Plant	Virus	Gene or Protein	Gene Expression control	Effects on Viral Infection	Known or Predicted Protein Function
Translation and replication	Arabidopsis	CLCV CMV TEV TuMV TMV	AtNSI ssi-2 1sp1 tom1 tom2A	Over expression Knock out Knock out Knock out Knock out	Enhanced infection efficiency Defective in TuMV and TEV replication Affects TMV RNA accumulation in protoplasts Affects TMV RNA accumulation in protoplasts	Acetyl transferase Plastid-localized stearoyl-ACP desaturase Translation initiation factor, eIF(iso)4E Essential constituent of replication complex Formation of RNA replication complex
Translation and replication	Nicotiana	TGMV	NbSCE1	Suppression	Suppresses TGMV replication	SUMO- conjugating enzyme
Cell-to-cell movement	Arabidopsis	CMV TCV	cum1-1 cum2-1	Knock out Knock out	Cell-to-cell movement of CMV is blocked Cell-to-cell movement of CMV is blocked	Translation initiation factor,eIF4E Translation initiation factor, eIF4G
Systemic movement	Arabidopsis	TMV	PME	Suppression	Defective in viral exit out of vascular system	Pectin methylesterase

CLCV, Cabbage leaf curl geminivirus; CMV, Cucumber mosaic virus; TEV, Tobacco etch virus; TGMV, Tomato golden mosaic virus; TMV, Tobacco mosaic virus; TuMV, Turnip mosaic virus (Kang B-C  et al. 2005. Annu Rev Phytopathol 43: 581-621)

of the resistance spectrum for a pair of alleles, genetic complementation must be formally assessed before different locus designations are accepted. Efforts are underway in many plant species and, to some extent, across the plant community to rationalize genetic nomenclature. Modern systems aim to reflect homology across sexually incompatible genera and the identity of the gene, where known. When multiple loci control the same virus or viral pathotype, the mode of inheritance of the resistance may be similar, as expected if the loci had arisen via duplicative processes that have generated the high degree of redundancy observed in plant genomes (e.g., 175), or the mode of inheritance may be different. One way whereby independent genes for resistance to the same pathogen can be distinguished may be the range of protection afforded by each allele. There are a number of examples of dominant and recessive genes that appear to

control a relatively wide range of viral genotypes that span multiple viral species, according to current delineation of viral taxa. The most dramatic examples appear to involve members of the Potyviridae, e.g., the I gene in Phaseolus vulgaris now appears to control a dominant resistance or a dominant necrotic response to ten different related potyviruses, Azuki mosaic virus, Bean common mosaic virus (BCMV), Bean necrotic mosaic virus, Blackeye cowpea mosaic virus, Cowpea aphid-borne mosaic virus, Passion fruit woodiness virus-K, Soybean mosaic virus

(SMV), Thailand Passiflora virus, Watermelon mosaic virus (WMV), and Zucchini yellow mosaic virus. Furthermore, this locus has been implicated in modulating a necrotic response to Bean severe mosaic virus, a member of the Comovirida. Recombination between any of the specificities listed above has never been observed despite more than 75 years of backcross breeding with this R gene, which has not been isolated, and independent sources of the I resistance allele show identical resistance spectra. Detailed physical mapping of the I locus has established that it occurs in a large cluster of TIR-NBS-LRR sequences.

Conversely, there are cases where resistance alleles at two or more loci are required to observe the resistant response. Because of the paramount agricultural importance of losses to BCMV, a well-known example is the bc-u system in Phaseolus vulgaris for resistance to a wide array of BCMV pathotypes. Resistance is observed only when the bc-u locus is homozygous recessive and one or more pathotype-specific genes, bc-1, bc-2, and bc-3, are also homozygous at one or more of three additional loci. In some cases, alleles at these loci affect pathotype specificity. In Capsicum, for example, full resistance is observed to another potyvirus, Pepper veinal mottle virus, only when the resistance alleles pvr12 (formerly pvr22) and pvr6 are homozygous. Here the pvr1 locus encodes an eIF4E homolog, and pvr6 is likely to encode eIF(iso)4E. Physical clustering of distinct R genes that control different pathotypes of the same viral species, closely related viral species, or diverse plant pathogen groups (e.g., viral, fungal, bacterial, or nematode pathogens) has also been widely noted and discussed in terms of R gene evolution and plant breeding. Two distinct types of gene clusters are clearly evident. One type of R gene cluster contains a set of genes, showing similar inheritance and resistance phenotypes that control very closely related viral genotypes. Presumably this type of cluster arose from the classic evolutionary trend of gene duplication, followed by divergence. This mechanism classically results in genes with related but slightly altered function. A notable example of this pattern occurs in Pisum sativum where recessive resistance has been mapped to two R gene clusters on linkage groups II and VI. In LG II, six very tightly linked monogenically inherited recessive loci (bcm, cyv1, mo, pmv, sbm2, and wmv) for resistance to BCMV, Bean yellow mosaic virus (BYMV), Clover yellow vein virus (ClYVV), Pea mosaic virus (PMV), Pea seed-borne mosaic virus (PSbMV-L1), and WMV, respectively, occur in a cluster but are separable by recombination. On LG VI, five distinct but very tightly linked loci have been identified that overlap with the specificities observed for the cluster on LG II. In this cluster, the loci cyv2, sbm1, sbm3, sbm4, and wlv confer resistance to ClYVV, PSbMV-P1, PSbMV-L1 or -P2, PSbMV-P4, and White lupin mosaic virus, respectively.

The second type of R gene cluster contains viral resistance along with R genes that control unrelated pathogens. These clusters may include a relatively large number of R genes and span megabases of genomic sequence. This type of R gene cluster occurs widely in monocots and dicots. For example, the wheat Bdv1 allele conferring resistance to Barley yellow dwarf virus (BYDV) is linked to fungal R genes Lr34 and Yr18. This type of cluster may, in fact, emerge as the most common genomic context for plant disease R genes, as more complete information about plant genome structure develops. Information regarding the content and distribution of R gene clusters is probably best understood in the dicot family, Solanaceae, the focus of major investments in genetic and genomic analyses. As tomato, potato, pepper, tobacco, and many minor solanaceous crops are affected by well-known viruses, extensive information is available regarding inheritance and mapping of viral R genes and many other R genes.

 A comprehensive genome-wide analysis of R gene clusters and their distribution within a series of crop genomes linked by comparative genetic mapping has been published for the Solanaceae. This study clearly demonstrated that R gene clusters often occur at homologous positions in related genomic regions, even in genera that diverged tens of millions of years ago. Furthermore, across genera, clusters contained either dominant R genes and QTL, or recessive genes and QTL, but not both dominant and recessive genes. These clusters may therefore consist of evolutionarily related sequences that diverged to control very different pathogen groups. When the sets of pathogens controlled by R genes in a given cluster were compared across taxa, no overlap of resistance specificities (i.e., the group of pathogen taxa controlled by R genes in the cluster) was initially observed, except in two cases on chromosomes 4 and 11. Both of these involved Phytophthora pathogens, P. capsici in pepper and P. infestans in potato. As additional R genes have been mapped, a striking pattern that includes viral R genes has now emerged (Table 1). On potato chromosome 12, the Rx1 gene conferring resistance to Potato virus X (PVX) is tightly linked to the Gpa2 locus for resistance to the cyst nematode Globodera pallida. This pair of specificities is also found very tightly linked in a second R gene cluster on potato chromosome 5. This cluster on chromosome 5 also contains resistance to P. infestans. When the inferred positions of all mapped R genes from tomato, potato, and pepper are collected on one comparative genetic map, at least five R gene groups can be discerned that contain a dominant R gene to Globodera and a dominant gene for resistance to Phytophthora, several of which also contain R genes or QTL for plant viruses including PVX, as mentioned above, TMV, CMV, Tomato yellow leaf curl virus (TYLCV), and potato virus Y.

A prediction, based on this observation, but not yet confirmed, is that despite these striking differences in pathogen specificity, the basic molecular structure of these genes will be generally similar, even between relatively distantly related host genera. This similarity may facilitate the molecular cloning and characterization of genes that reside in these clusters. Insofar as this prediction has been examined within a single R gene cluster in one plant taxon, e.g., Rx and Gpa2, it has been upheld. Dominant resistance is often, although not always, associated with the hypersensitive response (HR), possibly due to the frequent use of HR as a diagnostic indicator for field resistance by plant breeders. HR, induced by specific recognition of the virus, localizes virus spread by rapid programmed cell death surrounding the infection site, which results in visible necrotic local lesions. HR-mediated resistance is a common resistance mechanism for viruses and for other plant pathogens. Because the extent of visible HR may be affected by gene dosage, genetic background, environmental conditions such as temperature, and viral genotype, etc., schemes that classify or name virus R genes based on presence or absence of HR may obscure genetic relationships (see discussion of the Ry-mediated resistance to PVY in potato below). Over the past 10 years significant advances have been made in the understanding of the molecular basis of the HR-mediated resistance. More than 40 plant R genes showing monogenic dominant inheritance have been cloned. Several of these confer resistance to plant viruses. These include N for resistance to TMV in tobacco, Rx1 and Rx2 for resistance to PVX in potato, Sw5for resistance to Tomato spotted wilt virus (TSWV) in tomato, and RTM1, RTM2 to TMV, and HRT for resistance to Turnip crinkle virus (TCV), RCY1 for resistance to CMV, respectively, in Arabidopsis.

 In contrast to dominant R genes, many recessive R genes appear to function at the single cell level or affect cell-to-cell movement. More than half of the recessive R genes identified to date confer resistance to potyviruses, members of the largest and perhaps the most economically destructive family of plant viruses. This may be a consequence of some bias that affects the scope of our knowledge, or may be due to specific features of potyvirus biology. In general, considerably less is known regarding the mechanisms that account for recessively inherited resistance mechanisms. Several recessive R genes have recently been cloned and/or characterized including pvr1 (=pvr2), mo11, sbm1, and rym4/5 despite the possibility of bias affecting the comprehensiveness of available data, trends can be noted in the types of genetic resistance available to control viruses belonging to specific plant virus families. For example, resistance to CMV often shows complex inheritance. Very few monogenically inherited R genes are known, despite the enormous host range of this virus and its economic impact. Most resistance or tolerance of economic significance to this pathogen is quantitatively inherited. In contrast, resistance to tobamoviruses is widespread and is often monogenic dominant.

For some viral families of extreme agricultural importance, most notably the Geminiviridae, naturally occurring genetic resistance can be difficult to locate, and is often highly strain-specific and/or quantitatively inherited (i.e., each gene has a relatively slight positive effect on host response), making resistant varieties extremely difficult or impossible to develop without molecular markers and/or transgenic approaches. The Rx1 gene conferring resistance to Potato virus X (PVX) is tightly linked to the Gpa2 locus for resistance to the cyst nematode Globodera pallida. This pair of specificities is also found very tightly linked in a second R gene cluster on potato chromosome 5. This cluster on chromosome 5 also contains resistance to P. infestans. When the inferred positions of all mapped R genes from tomato, potato, and pepper are collected on one comparative genetic map, at least five R gene groups can be discerned that contain a dominant R gene to Globodera and a dominant gene for resistance to Phytophthora, several of which also contain R genes or QTL for plant viruses including PVX, as mentioned above, TMV, CMV, Tomato yellow leaf curl virus (TYLCV), and potato virus Y. A prediction, based on this observation, but not yet confirmed, is that despite these striking differences in pathogen specificity, the basic molecular structure of these genes will be generally similar, even between relatively distantly related host genera. This similarity may facilitate the molecular cloning and characterization of genes that reside in these clusters. Insofar as this prediction has been examined within a single R gene cluster in one plant taxon, e.g., Rx and Gpa2, it has been upheld.

Dominant resistance is often, although not always, associated with the hypersensitive response (HR), possibly due to the frequent use of HR as a diagnostic indicator for field resistance by plant breeders. HR, induced by specific recognition of the virus, localizes virus spread by rapid programmed cell death surrounding the infection site, which results in visible necrotic local lesions. HR-mediated resistance is a common resistance mechanism for viruses and for other plant pathogens. Because the extent of visible HR may be affected by gene dosage, genetic background, environmental conditions such as temperature, and viral genotype, etc., schemes that classify or name virus R genes based on presence or absence of HR may obscure genetic relationships (see discussion of the Ry-mediated resistance to PVY in potato below).

 Over the past 10 years significant advances have been made in the understanding of the molecular basis of the HR-mediated resistance. More than 40 plant R genes showing monogenic dominant inheritance have been cloned. Several of these confer resistance to plant viruses. These include N for resistance to TMV in tobacco, Rx1 and Rx2 for resistance to PVX in potato, Sw5 for resistance to Tomato spotted wilt virus (TSWV) in tomato, and RTM1, RTM2 to TMV, and HRT for resistance to Turnip crinkle virus (TCV), RCY1 for resistance to CMV, respectively, in Arabidopsis. In contrast to dominant R genes, many recessive R genes appear to function at the single cell level or affect cell-to-cell movement. More than half of the recessive R genes identified to date confer resistance to potyviruses, members of the largest and perhaps the most economically destructive family of plant viruses. This may be a consequence of some bias that affects the scope of our knowledge, or may be due to specific features of potyvirus biology. In general, considerably less is known regarding the mechanisms that account for recessively inherited resistance mechanisms. Several recessive R genes have recently been cloned and/or characterized including pvr1 (=pvr2), mo11, sbm1, and rym4/5.

 Despite the possibility of bias affecting the comprehensiveness of available data, trends can be noted in the types of genetic resistance available to control viruses belonging to specific plant virus families. For example, resistance to CMV often shows complex inheritance. Very few monogenically inherited R genes are known, despite the enormous host range of this virus and its economic impact. Most resistance or tolerance of economic significance to this pathogen is quantitatively inherited. In contrast, resistance to tobamoviruses is widespread and is often monogenic dominant. For some viral families of extreme agricultural importance, most notably the Geminiviridae, naturally occurring genetic resistance can be difficult to locate, and is often highly strain-specific and/or quantitatively inherited (i.e., each gene has a relatively slight positive effect on host response), making resistant varieties extremely difficult or impossible to develop without molecular markers and/or transgenic approaches.