Characteristics and phenotype of resistance to S. hermonthica
Rice cultivars revealed striking differences in their ability to support the growth and development of S. hermonthica. A large number of rice cultivars (O. sativa subspecies japonica and indica, O. glaberrima and wild relatives of rice) were screened initially for their postattachment resistance to S. hermonthica; all were found to be susceptible, to varying degrees, with the exception of one cultivar, Nipponbare (japonica-type), which exhibited strong resistance.
In a susceptible interaction (IAC 165 or Kasalath) the parasite haustorium attached to the root system within 1 d of placing pregerminated seeds onto the roots. The initial haustorium forms in response to haustorial-inducing factors present in host root exudates (Estabrook & Yoder, 1998; Yoder, 1999; 2001). By day 3, the endophyte had penetrated both the root cortex (a process that is also responsive to host-specific factors) and the root endodermis, and the first parasite–host xylem–xylem connections were just visible (Fig. 1a,i and Fig. 2). In order for the parasite to form xylem–xylem connections, the endophye must penetrate the host endodermis and make direct contact with the host stele (Yoder, 1997), although little is known about the host factors required for successful penetration of the endodermis. Once xylem–xylem connections had formed, the parasites developed rapidly; by day 9, leaf primordia were well developed and the haustorial tissues had begun to differentiate (Fig. 1a,ii). A cross-section through the parasite haustorium at 21 dai revealed well-developed parasite–host xylem–xylem connections and a clearly differentiated vascular core and hyaline body (Fig. 1a,iii). A mature haustorium is essential for parasite growth and survival. First, xylem–xylem connections allow the movement of solutes from host to parasite and, second, the hyaline body is thought to metabolise these solutes, or at least to regulate the supply of nutrients to the developing parasite. The tissues of the hyaline body demonstrate intense metabolic activity and many of the enzymes involved in N metabolism are located in this region (Taylor, 2001).
Figure 1. (a) Transverse sections of embedded tissue of susceptible (Kasalath) (I–iii) and resistant (Nipponbare) (iv–vi) rice roots 3, 9 and 21 d after inoculation with Striga hermonthica. The scale bar represents 0.1 mm. En, endophyte (internal part of haustorium); Hc, host root cortex; He, host endodermis; Hx, host xylem; Hx–Px, host–parasite xylem continuity; Hy, hyaline body; P, parasite haustorium; and Px, parasite xylem vessels. (b) The root systems of susceptible (IAC 165 and Kasalath) and resistant (Nipponbare) rice cultivars 25 d after infection with the angiosperm parasite Striga hermonthica.
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Figure 2. Development of Striga hermonthica on the roots of IAC 165, Kasalath and Nipponbare 3, 9, 15 and 21 d after inoculation. Stages of development of S. hermonthica were defined as: S1, S. hermonthica attached, seed coat is intact and a tubercle is evident; S2, emergence of leaf primordia; S3, S. hermonthica shoots have three to five scale leaf pairs; S4, S. hermonthica shoots have six or more leaf pairs; Dead, S. hermonthica plants attached and died.
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Although both IAC 165 and Kasalath supported Striga development, there were clear differences in the rate of growth of the parasites; those on IAC 165 developed more quickly and were larger than those on Kasalath (Figs 1b and 2). Twenty-one days after inoculation, 89% of parasites attached to IAC 165 had between three and six scale leaf pairs (stages S3 and S4) and fewer than 1% of parasites were dead (Fig. 2). By contrast, only 36% of parasites attached to Kasalath had reached developmental stages S3 or S4 at this time, and 11% of parasites were dead. The difference in growth rate and developmental stage of the parasites may be the result of differences in the ability of the hosts to supply nutrients to the parasites, but it is more likely to reflect genotypic differences in the susceptibility/resistance of the two cultivars, with Kasalath exhibiting some resistance to the parasite.
The early stages of parasite development on Nipponbare were similar to those on Kasalath or IAC 165; parasites attached to the host root system within 1 d of inoculation and, by day 3, the parasitic endophyte had successfully penetrated the host root cortex, thus demonstrating that host-specific factors necessary for early haustorial formation and successful penetration of the cortex were present in this cultivar. At this early stage, the external appearance of the parasites on Nipponbare resembled those on susceptible varieties (Fig. 1a,i and iv); there was no evidence of a hypersensitive reaction, as observed in the sorghum cultivar Framida following infection with S. asiatica (Mohamed et al., 2003). However, in contrast to Kasalath or IAC 165, the parasite rarely breached the endodermis in Nipponbare roots. The reason for this is unclear as there was no obvious difference between the endodermis of Nipponbare and that of susceptible cultivars; for example, it did not appear to be more heavily lignified (Fig. 1a). By 9 dai it was clear that the parasite was unable to form parasite–host xylem–xylem connections; in many cases, the endophyte passed straight through the root cortex and emerged from the other side of the root, or it encircled the vascular core of the host within the root cortex (Fig. 1a,iv–vi). Vascular continuity between host and parasite not only allows the transport of water and nutrients from host to parasite, but may also provide factors required for further differentiation of the haustorium. The haustorium of parasites attached to Nipponbare did not mature and differentiate (Fig. 1a,vi). Thus, by day 21, 49% of the attached parasites were either dead or showed signs of necrosis (Fig. 1a,vi inset, Fig. 1b and Fig. 2). Only 2% of attachments developed beyond the primordia leaf stage, and stem elongation was rare.
It is clear that Nipponbare is resistant to S. hermonthica, at least in part because the parasite failed to form vascular continuity with the host. This type of resistance differed from that observed in the sorghum cultivars Framida and Dobbs where, following attachment of S. hermonthica, a proportion of the parasites died owing to the onset of a rapid hypersensitive reaction (Mohamed et al., 2003) and also in the nonhost interaction between S. asiatica and marigold. In the latter, penetration of the cortex was terminated early and the endophyte rarely reached the endodermis (Hood et al. 1998). Gowda et al. (1999) used differential display techniques to identify host genes expressed in marigold roots in response to penetration by S. asiatica. These authors isolated and cloned a novel gene (NRSA-1) that had homology to known plant disease-resistance genes, but which differed structurally from those previously described. A heavy deposit of lignin was often also observed around the invading endophyte, and haustorial cells at the host–parasite interface showed signs of necrosis. Recently, resistance to S. hermonthica has also been identified in T. dactyloides, a wild relative of maize (Gurney et al., 2003). In this case, S. hermonthica arrested after host–parasite xylem–xylem continuity was established. The haustorium failed to differentiate either because the host lacked signals required for haustorial development or, more likely, because T. dactyloides produced a signal that inhibited haustorial development. The resistance observed in Nipponbare is most similar to that observed in the resistant interaction between vetch Vicia atropupurea cultivar Popany and the angiosperm parasite, Orobanche aegyptiaca. In this interaction, the parasite haustorium penetrated the root, but was blocked at the root endodermis layer (Goldwasser et al., 2000). However, in contrast to Nipponbare, the blockage was coupled with a large secretion of unknown composition, which prevented the parasite from establishing vascular continuity.
Nipponbare is resistant to S. hermonthica as the parasite fails to form xylem–xylem connections, thus preventing the transport of water, nutrients and developmental cues that allow successful development of the parasite. The most common resistance mechanisms are those in which the host lacks factors needed by the parasite, particularly germination stimulants (Parker & Riches, 1993), but this is the first example of resistance caused by a lack of formation of parasite–host vascular continuity. The majority of resistance/tolerance factors are not simply inherited, as illustrated by a recent study of the genomic regions influencing low germination stimulant production (and hence resistance/tolerance to S. hermonthica) in two recombinant inbred populations of sorghum (Haussmann et al., 2004). Such studies suggest that resistance is polygenic in nature and hence extremely important in an agricultural context. In order to begin to elucidate the genetic basis of resistance in Nipponbare, a QTL analysis was undertaken utilizing a mapping population of BILs (Nipponbare/Kasalath//Nipponbare).
The genetic basis of resistance to S. hermonthica
The distribution of S. hermonthica resistance in the mapping population is consistent with a polygenic mode of inheritance (Fig. 3). The mean resistance in the mapping population was intermediate between that observed in the parental lines, although the distribution of resistance in the mapping population did show slight bimodality, which may be consistent with some genes of major effect segregating. The range in resistance in the mapping population varied from more susceptible than the most susceptible Kasalath plant to more resistant than the Nipponbare parental line (Fig. 3).
Figure 3. Distribution of resistance to Striga hermonthica in the mapping population, F1BC14 (grey bars), and in the parental lines Kasalath (white bars) and Nipponbare (black bars). n = 16 for parental lines; n = 391 for the mapping population.
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Seven QTL were identified at the genome-wide P < 0.10 level (Table 2, Fig. 4), four of which provided test statistics that were never exceeded in 1000 permutations (genome-wide P < 0.001). Unsurprisingly, the Nipponbare allele conferred greater resistance than the Kasalath allele at six of the seven QTL. However, a QTL of large effect on chromosome 4 showed a contrasting effect; the Kasalath allele was more resistant than the Nipponbare allele. In practical terms this discovery is exciting; introgression of the Kasalath allele into a Nipponbare background would result in a phenotype even more resistant than Nipponbare. Consistent with this is the observation that a small number of BILs exhibited greater resistance to S. hermonthica than Nipponbare (Fig. 3).
Table 2. Striga resistance quantitative trait loci (QTL) significant at the genome-wide threshold (P < 0.10)
|Chromosome||Position||LRT||P||PVE||Allelic substitution (standard deviations)||Confirmed|
|1||46 cm||17.74||< 0.001*||1.8|| 0.036 (0.45)||No|
|4||79 cm||66.13||< 0.001***||7.6||−0.064 (−0.8)||Yes, P < 0.0014|
|5||77 cm||19.65||< 0.001*||1.9|| 0.039 (0.49)||Yes, P < 0.0014|
|6||97 cm||45.04||< 0.001***||4.2|| 0.051 (0.64)||Yes, P < 0.0014|
|7||74 cm||57.56||< 0.001***||5.5|| 0.060 (0.75)||No|
|8||32 cm||21.59||< 0.001**||2.1|| 0.038 (0.48)||No, P = 0.004|
|12||41 cm||63.28||< 0.001***||7.4|| 0.075 (0.94)||Yes, P < 0.0014|
Figure 4. Comparison of the estimates of genome-wide quantitative trait loci (QTL) additive effects for the initial (unbroken black line) and confirmatory (dotted line) screens. Estimates of QTL additive effects on host resistance to Striga hermonthica (arcsine transformed) are plotted for the entire rice genome (chromosomes 1–12), for the initial screen (black curve) and for the confirmatory screen (dotted curve). Additive effects of > 0 are consistent with the Nipponbare allele conferring resistance. The test statistics at each location are significantly positively correlated between screens (r = 0.660; P = 0.0012), indicating that the findings of the first screen are broadly confirmed by the findings of the second screen. Note that the apparent larger effect sizes observed in the confirmatory screen are probably attributable to an effect size overestimate caused by a relatively small sample size (n = 196). Vertical lines indicate a chromosome boundary. Positions of the seven QTL identified in the initial screen are shown. *Genome-wide significant at P < 0.1; **genome-wide significant at P < 0.05; ***genome-wide significant at P < 0.001.
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The magnitude of the QTL effects varied from 1.8% to 7.6% of the variance in mapping population resistance, and between them accounted for 31% of the overall variance. An alternative, and for breeding purposes more relevant, method of measuring QTL magnitude is as an effect size relative to the phenotypic variance observed in the parental races (Orr, 2001). Here, resistance in both Nipponbare and Kasalath had a standard deviation of approx. 0.08 (Table 3), while the effects of an allelic substitution at the QTL ranged from 0.036 to 0.075 (Table 2). Thus, under the usual definition of an allelic substitution that alters phenotype by at least 0.5 of a phenotypic standard deviation, these QTL should be considered major genes (Falconer & Mackay, 1996). The QTL on chromosomes 4 and 12 had the largest effect on S. hermonthica resistance; allelic substitutions at these two QTL approached 1 standard deviation (SD), and between them they explained 15% of the variance in resistance.
Table 3. Mean resistance of the mapping populations and the parental lines
|Population||n||Mean resistance (SD)|
|Kasalath|| 16||0.740 (0.080)|
|Nipponbare|| 16||1.188 (0.078)|
|Mapping population||391||0.986 (0.185)|
|Confirmatory population||196||1.053 (0.210)|
We took the unusual step of performing a confirmatory genome scan for QTL. Four of the seven QTL were detected in the second scan (at nominal significance P < 0.0014), and can therefore be regarded as confirmed linkage (Lander & Kruglyak, 1995). The QTL on chromosomes 1, 7 and 8 were not confirmed. The genome-wide test statistics were highly and significantly correlated between mapping experiments (r = 0.660, P = 0.0012, Fig. 4), indicating that the results from the first experiment were largely replicated in the confirmatory screen. Note the striking similarity between the genome-wide estimates of QTL effect in the two screens (Fig. 4). At some QTL (chromosomes 4, 5, 8 and 12), the confirmatory screen effect size was larger than that reported for the original screen. However, we report effect sizes as those estimated from the first screen because effect sizes may be upwardly biased in the smaller confirmatory screen (Beavis, 1994).
A key challenge in the future is to fine map, and ultimately to identify, the causative mutations or quantitative trait nucleotides (QTN) that are responsible for the S. hermonthica QTL. We aim to use an integrated microarray and fine-mapping approach to achieve these goals. Genes that show differential expression in infected tissues of the two parental races, and that map closely to the QTL, are excellent functional candidates for host-resistance genes (Beavis, 1994; Schadt et al., 2003). Fine mapping of the QTL can be achieved by the creation of nearly isogenic lines (NILs) and the subsequent scoring of resistance within those lines. Recent genetic tools for indica and japonica rice varieties include a large database of single nucleotide polymorphisms (Feltus et al., 2004), first draft genome sequences (Goff et al., 2002; Yu et al., 2002) and microarray technology. Thus, future prospects for the discovery of S. hermonthica resistance QTN are encouraging. Furthermore, the high degree of conserved synteny exhibited between many plant species (Salse et al., 2002) means that QTL identified in this system may rapidly be investigated in the most economically important crops in sub-Saharan Africa.