Characterisation of the effects of a major QTL of the partial resistance to Rice yellow mottle virus using a near-isogenic–line approach

Physiological and Molecular Plant Pathology 63 (2003) 213–221 www.elsevier.com/locate/pmpp

Characterisation of the effects of a major QTL of the partial resistance to Rice yellow mottle virus using a near-isogenic –line approach D. Ioannidoua, A. Pinela, C. Brugidoua, L. Albara, N. Ahmadib, A. Ghesquierea, M. Nicolea, D. Fargettea,* a

IRD, BP 64501, 34394 Montpellier cedex 5, France b CIRAD, 34398 Montpellier cedex 5, France Accepted 19 December 2003

Abstract Two quantitative trait loci (QTL12 and QTL7) acting in epistatis and associated with Rice yellow mottle virus (RYMV) infection were introgressed from the traditional Oryza sativa japonica cultivar Azucena into the improved indica cultivar IR64. The response to RYMV infection was followed over time in this nearly isogenic line (NIL). In leaves, the virus coat protein content was detected by ELISA and RNA by RT-PCR. In tissues, the coat protein was localised by immuno-fluorescence labelling and RNA by in situ hybridisation. We found (i) that Azucena combined a tolerance—characterised by mild symptoms despite generalised distribution and large accumulation of the virus—and a partial resistance, (ii)that partial resistance under the control of QTL12 and QTL7, dissociated in the NIL from tolerance and any other morphological or physiological Azucena traits, was expressed similarly in a japonica and in an indica genetic background, (iii) that partial resistance was transient and consisted in a 1-week delay in virus accumulation and symptom expression; passed this delay, partial resistance brokedown, and (iv) a similar delay in virus detection was observed at tissue levels, in the vascular bundle-sheath layers. Impaired virus movement caused by the absence or mutation of a QTL12-encoded plant factor necessary for virus transport through vascular bundle sheaths is proposed to explain this partial resistance. q 2004 Elsevier Ltd. All rights reserved. Keywords: Rice yellow mottle virus; Quantitative trait locus; Partial resistance; Tolerance; Oryza sativa; Vascular bundle sheath; Nearly isogenic line

1. Introduction Rice yellow mottle virus (RYMV), of the genus Sobemovirus [27], causes a major disease of rice in Africa [3]. RYMV is transmitted by chrysomelid beetles and other biotic and abiotic means [2,31]. Infected plants show mottling and yellowing of the leaves, stunting, partial emergence of the panicles and sterility [10]. Almost total yield losses have been reported in Oryza sativa indica cultivars including the highly productive IR64 [1]. Since RYMV management through vector control and cultural practices is only partially effective [1], the development of resistant cultivars is an important requirement in ricebreeding programmes in Africa. Today, three types of varietal resistance to RYMV are known: a high resistance [8,24], a partial natural resistance [17], * Corresponding author. Tel.: þ 33-467-416-227; fax: þ33-467-416-330. E-mail address: [email protected] (D. Fargette). 0885-5765/$ - see front matter q 2004 Elsevier Ltd. All rights reserved. doi:10.1016/j.pmpp.2003.12.005

and a resistance obtained through genetic transformation [29]. Varieties with partial resistance to RYMV all belong to the tropical japonica sub-species and are not adapted to lowland cultivation. The genetic determinism of this resistance is polygenic [5]. Resistance quantitative trait loci (QTL) have been mapped in a double-haploid population of a cross between a susceptible lowland indica cultivar, IR64, and a resistant upland japonica cultivar, Azucena [4,6,17,30]. Fifteen QTLs have been detected on seven chromosomal fragments. A colocalisation was observed between some resistance QTLs and QTLs involved in morphological and growth characteristics. However, one that mapped on chromosome 12 (QTL12) was independent from morphological traits and was associated more specifically with low virus content and symptom expression in systemically infected leaves [7]. Moreover, an epistatic interaction was found between the QTL12 and a region of chromosome 7 (QTL7). This interaction explained ca. 36% of the quantitative variation of virus content in the IR64 £ Azucena DH population [30]. Accordingly, QTL12 and QTL7

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from Azucena were introgressed into IR64 through cycles of molecular marker-assisted back-crosses. Ahmadi et al. [4] confirmed experimentally the complementary epistasis genetic model with a nearly isogenic line (NIL) carrying over 90% of the recipient IR64 genome. At early stages of infection, the response to RYMV infection in Azucena, compared to IR64, was characterised by a low virus titer. At later stages of infection, infected Azucena expressed mild symptoms despite a generalised distribution and a high virus accumulation [20]. Accordingly, it was hypothesized that the dual response of Azucena combined a partial resistance at early stages of infection and a tolerance apparent at later stages. However, putative resistance and tolerance must be dissociated genetically to validate this hypothesis and to characterise their respective phenotype independently. We used the NIL in order to dissociate the effects of partial resistance from tolerance, and to characterise, at the plant, leaf and tissue levels, the phenotype associated to the presence of the QTL12. The response to RYMV infection was followed over time. In leaves, symptoms were recorded, the virus coat protein (CP) content was assessed by ELISA and the RNA by RT-PCR, whereas in tissues the CP was localised by immunofluorescence labelling and the RNA was revealed by in situ hybridisation.

2. Materials and methods 2.1. Plant material Among the DH population derived from a cross between the cultivars IR64 (susceptible O. sativa indica) and Azucena (partially resistant O. sativa japonica), line P303 was selected because of: (i) its high level of resistance, (ii) the presence of the allele from Azucena at the RG869 (QTL12) and CDO418 (QTL7) loci, (iii) recombinations on each side of the QTL12 near the RG869 locus, and (iv) a predominant IR64 ‘background’ of 60%. Details of the introgression and marker-assisted selection are given in Ref. [4]. Briefly, DH line P303 was retro-crossed twice with the IR64 parent by a selfed generation BC1F2. A plant with Azucena alleles homozygous for QTL12 and QTL7 was selected and markerassisted selection was performed to eliminate other resistance QTLs from Azucena. A second round of selfing was made to increase the seed set and to produce the BC1F4 genetic material used to assess resistance. Theoretically, the resulting genetic material accounted for 90% of the IR64 genetic background, but taking into account the marker-assisted selection against the other QTLs, it could be considered as IR64-isogenic for RYMV resistance genes except for QTL12 and QTL7. In this study, BCF1F4 NIL line was compared to the IR64 and Azucena parents. The highly resistant cultivar

Gigante, with a different and monogenic recessive genetic determinism [6], was introduced as control. 2.2. Virus inoculation An isolate of RYMV which induced the most commonly encountered pathogeny pattern in cultivars IR64, Azucena and Gigante was chosen for study. Plants were grown in a glasshouse under controlled conditions (28–32 8C, 13 h illumination at 120 mE m22 s21 of PAR and 90% humidity). Inoculations were made 2 weeks after planting on the third expanded leaf (leaf III on the numbering system of Matsuo and Hoshikawa [23]). The inoculum was obtained by grinding 1 g of leaf in 10 ml of inoculation buffer (0.1 M KH2PO4 and 0.1 M Na2HPO4, adjusted to pH 7.2). Inoculation was performed by rubbing the leaf with inoculum mixed with carborundum. In these conditions, during seven experiments, plant growth and symptom expression were followed with ca. 60 plants of each line. In each experiment, the group of 60 plants responded homogeneously to virus infection. 2.3. ELISA and PCR tests ELISA tests were performed as described in Ref. [17]. The plant material was stored at 2 80 8C. Leaves were ground in PBS-T (1 g/10 ml). The extracts were centrifuged at low speed (5 min at 10,000g) and the DAS-ELISA tests were performed on the supernatant. A volume of 100 ml of polyclonal antibodies of the RYMV-Mg antiserum diluted 1:1000 in coating buffer was incubated for 2 h at 37 8C in each well. A blocking step with 200 ml of 3% skimmed milk in PBS-T was performed. Volumes of 100 ml of leaf extract diluted 1:1, 1:10 and 1:100 and RYMV-Mg antiserum conjugated with alkaline phosphatase diluted 1:1000 were incubated for 2 h at 37 8C. A volume of 100 ml of paranitrophenyl phosphate in diethanolamine buffer was loaded in each well. Absorbance was measured at 405 nm (Dynatech MR 5000) after different incubation periods at room temperature. RT-PCR tests were performed as described in Ref. [20]. 2.4. Immuno-fluorescence tests The experiments were performed as described in Ref. [20]. Fragments (ca. 2 mm long) of inoculated, systemically infected and healthy leaf tissues were fixed for 3 h in 1% (v/v) glutaraldehyde 4% and (w/v) paraformaldehyde, and dehydrated using a graded series of ethanol before embedding in LR White (TAAB England). Semi-thin sections (1.5 mm) made on a Reichert Ultracut E microtome were immuno-stained as described in Ref. [20]. The experiment was performed twice using two plants per treatment. Two blocks were treated and at least three semithin sections per block were observed.

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2.5. In situ hybridisation tests In situ hybridisation of the RNA in rice tissues was performed according to the method of Kronenberg et al. [22] with modifications in the fixation buffer [9]. Briefly, pieces of tissues were embedded in a plastic resin (methacrylate/ mixture of n-butyl methacrylate and methyl methacrylate), sectioned (2.5 mm) and mounted on microscopic slides treated with biobond 2%. Afterwards, the sections were treated with proteinase K (0.5 mg/ml). After stopping proteinase K reaction, sections were dehydrated and incubated at 37 8C overnight in a hybridisation buffer. The probe was double-stranded DNA obtained from the fulllength genome of an RYMV-CI clone [12], from the plasmid pUC 19, labelled randomly with digoxigenin-UTP. The probe was detected using an anti-DIG conjugate at 1:250 and NBT/BCIP substrate (Starter Kit, Boehringer). The signal was completed after 45 min. The experiment was made twice using two plants per treatment. Two blocks were treated and at least three semi-thin sections per block were observed. Inoculated and systemically infected leaves were tested. The specificity of the in situ hybridisation was demonstrated by omitting probe or antibody in the detection procedure which led to the complete absence of any purple precipitate on the sections.

3. Results 3.1. Plant and leaf symptoms The morphology and growth pattern of IR64 and the NIL were very similar. This confirmed the elimination of most of the QTLs/genes from Azucena involved in morphology and plant height such as the Azucena wild-type allele of the semi-dwarf gene sd1 on chromosome 1. At 5 days postinoculation (DPI), no virus symptoms were apparent in any of the tested lines. At 9 DPI, mottle developed on leaves of IR64, but only sparse dots were distinguished on leaves of the NIL or of Azucena (data not shown). At 14 DPI, IR64 plants turned yellow (Fig. 1), whereas leaves developed a severe mottling (Fig. 2). By contrast, Azucena plants showed no substantial reduction of plant growth and leaves developed a light mottle only. At 14 DPI, the NIL responded like Azucena with no plant stunting and light leaf symptoms. At 21 DPI, IR64 plants became stunted and a generalised necrosis developed on leaves. In Azucena, plant size remained unaffected and leaves developed leaf mottling. At 21 DPI, the NIL responded like the susceptible IR64 with plant yellowing, stunting, leaf mottling and necrosis. At 28 DPI, a generalised necrosis developed on IR64 and on the NIL leading to plant death, whereas Azucena growth remained unaffected and leaf symptoms limited to mottle (Fig. 1). Symptoms were not observed in Gigante at any date. This pattern of symptom expression was observed in five of the seven independent replicate

Fig. 1. Healthy (H) and infected (I) plants of the indica cultivar IR64 (left), of the IR64-nearly isogenic line (NIL) carrying the major QTL (QTL12) of the partial resistance introgressed from the japonica cultivar Azucena (centre), and Azucena (right), 14 (top), 21 (middle) and 28 (bottom) days post-inoculation (DPI).

experiments, and confirmed with the NILs at more advanced stages of isogenisation obtained after two additional backcrosses. In the other two experiments, the differences between the NIL and IR64 at early stages of infection were less pronounced. 3.2. Virus accumulation in plants Accumulation of the viral CP in the three cultivars and the NIL was assessed by ELISA, and the results were compared through variance analysis using Systat 5 software [34]. There were significant cultivar (P , 0:0001; df ¼ 3,

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Fig. 2. Leaf symptoms on the indica cultivar IR64 (left), on the IR64-nearly isogenic line (NIL) carrying the major QTL (QTL12) of the partial resistance introgressed from the japonica cultivar Azucena (centre), and on Azucena (right), 14 (top) and 21 (bottom) days post-inoculation (DPI).

F ¼ 300), date (P , 0:0001; df ¼ 3, F ¼ 394) and leaf effects (P ¼ 0:01; df ¼ 1, F ¼ 7). Notably, the relative responses of cultivars IR64, Azucena and the NIL differed markedly among dates (Fig. 3). In the inoculated leaves (leaf III), CP detection was erratic 5 DPI (data not shown). No significant differences between the lines were apparent 9 DPI (Fig. 3). By contrast, at 14 DPI, Azucena and the NIL responded similarly with CP contents significantly lower than those of IR64. At 21 DPI, there was no significant difference between IR64 and the NIL. Assessment in the systemically infected leaves (leaf V) was more discriminant than in the inoculated leaves at early stages of infection. At 9 DPI, the NIL and Azucena had virus contents lower than IR64 (Fig. 3). At 14 DPI, the virus content in the NIL was intermediate between those of IR64 and Azucena, suggesting a resistance level of the NIL somewhat less than in Azucena. At 21 DPI, there were no differences among the lines. Throughout the experiment, virus was not detected in Gigante in either inoculated or systemically infected leaves.

PCR detection in the systemically infected leaves allowed a more discriminant comparison at early stages of infection (Table 1). At 5 DPI, virus was detected in IR64, but not in the NIL. At 9 DPI, there was a clear distinction between the NIL and Azucena on one hand, and IR64 on the other. At 14 DPI, there was no difference among the lines, possibly because of a ‘saturation’ of the PCR response due to a high viral RNA content of the plants at this date. By contrast, PCR on the inoculated leaf did not discriminate among lines, as there was similar detection among the lines, even at the early stages of experiment. This may have resulted from amplification of residual RNA on the leaf surface after mechanical inoculation. Differences in virus detection (CP and RNA) among lines paralleled differences in symptom expression at early stages of infection. At later stages, virus titre and symptom severity were unrelated; high virus content being associated with conspicuous symptoms in IR64 and the NIL, and with relatively inconspicuous symptoms in Azucena. Overall, the NIL, with a morphology similar to IR64, responded as Azucena at the early stage of infection (# 14 DPI) with milder symptom and lower virus content than IR64. One week later, the NIL responded like IR64 with necrotic symptoms, whereas symptoms in Azucena remained mild. This indicates that the partial resistance associated to QTL12 and QTL7 was transferred successfully in the NIL, was expressed during the first 2 weeks after inoculation, and brokedown later. Complementary experiments were conducted to confirm that partial resistance had a genetic determinism distinct from tolerance. An isolate known to induce severe symptoms on Azucena was inoculated to the NIL and to Azucena in comparison with the reference isolate. As expected, tolerance in Azucena was apparent against the reference isolate, but was overcome by the severe isolate. By contrast, partial resistance in the NILs was expressed similarly after inoculation by either isolate with the same delay in symptom expression followed by the same resistance breakdown. This differential response confirmed that partial resistance is genetically distinct from tolerance. The phenotype of partial resistance in the NIL was studied at tissue levels independently from tolerance and any other morphological or physiological Azucena traits. 3.3. Virus localisation in tissues Observations of transverse sections distinguished four groups of cell layers [15]: (i) the epidermal layers (higher and lower epidermis), (ii) non-vascular tissues (sclerenchyma and mesophyll tissues), (iii) the two bundle sheaths (the vascular bundle sheath and the inner mestome sheath, characteristics of C3 gramineaceous species), and (iv) vascular elements (xylem vessels and parenchyma, sieve tubes and phloem parenchyma). CP and RNA detection in inoculated leaves were more erratic and less discriminant

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Fig. 3. Virus accumulation in the indica cultivars Gigante and IR64, in the japonica cultivar Azucena, and in the IR64-nearly isogenic line (NIL) carrying the major QTL (QTL12) of the partial resistance introgressed from Azucena, 9, 14 and 21 days post-inoculation assessed in ELISA tests through absorbances (A 405 nm) of extracts of the inoculated leaf (leaf III; top) and of the first systemically infected leaf (leaf V; bottom). Different letters indicate statistically different responses (at the 5% level) in multiple means comparisons.

than in systemically infected leaves. Stress from the mechanical inoculation resulted in numerous wounds, altered the tissue response and induced an early necrosis of the inoculated leaves. Furthermore, challenge with a massive inoculum masked potential differences in response between susceptible and resistant cultivars. Thus, only results obtained with systemically infected leaves were informative and presented. The CP and the RNA were not detected in epidermal layers, in the sclerenchyma and in the xylem vessels of any lines at any time after inoculation (Table 2). In the mesophyll and in the vascular bundle sheath, the RNA was detected more consistently than the CP. In sieve tubes and phloem parenchyma, both RNA and CP detections were different among replicates and among dates, likely reflecting whether virus trafficked into or from vascular tissues. CP and RNA were never detected in tissues of Gigante. Ultimately, the same tissues of IR64, Azucena and the NIL were infected but the timing and the intensity of detection did differ among them. In the susceptible tissues, generalised RNA detection was apparent 14 DPI in IR64, but only 1 week later, at 21

DPI, in Azucena. All together, at the tissue level, the NIL responded as Azucena, as determined by in situ hybridisation tests and immuno-fluorescence tests (Table 2). In the NIL, as in Azucena, RNA detection was weak and restricted at 14 DPI, whereas there was a generalised and intense detection in the susceptible tissues

Table 1 PCR detection of RYMV in inoculated and systemically infected leaves of lines IR64, NIL-BC1F4 and Azucena 5, 9 and 14 DPI Lines

IR64 NIL Azucena

Inoculated leaf

Systemically infected leaf

5 DPI

9 DPI

14 DPI

5 DPI

9 DPI

14 DPI

2 2 2

1 0/1 1

3 3 3

2 0 nea

3 0/1 0/1

3 3 3

PCR amplification was coded as followed: ‘0’ absence of band; ‘0/1’ a weak band and differences of response among replicates; ‘1, 2, 3’ bands with increasing intensity. a Leaf not emerged.

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Table 2 Coat protein localisation by immuno-fluorescence and RNA localisation by in situ hybridisation in tissues of systemically infected leaves ’V’ of IR64, NIL and Azucena 9, 14 and 21 DPI Detection

Epidermal layers Higher epidermis Lower epidermis Non-vascular tissues Sclerenchyma Mesophyll Bundle sheaths Vascular bundle sheath Mestome Vascular elements Xylem vessels Xylem parenchyma Sieve tubes Phloem parenchyma

IR64

NIL

Azucena

9 DPI

14 DPI

21 DPI

9 DPI

14 DPI

21 DPI

9 DPI

14 DPI

21 DPI

CPa RNAb CP RNA

0 0 0 0

0 0 0 0

0 0 0 0

0 0 0 0

0 0 0 0

0 0 0 0

0 0 0 0

0 0 0 0

0 0 0 0

CP RNA CP RNA

0 0 0 3

0 0 0 3

0 0 2 3

0 0 0 0

0 0 0 3

0 0 0 3

0 0 0 2

0 0 0 2

0 0 0 3

CP RNA CP RNA

0 1 0 0

0 1 1 2

0 1 2 1

0 0 0 0

0 0 0 0

0 2 2 2

0 0 0 0

0 0 0 0

0 2 1 2

CP RNA CP RNA CP RNA CP RNA

0 0 1 1 0 2 1 3

0 0 2 3 2 0 3 0

0 0 3 2 1 2 3 2

0 0 0 0 1 0 1 0

0 0 2 2 2 0 3 2

0 0 3 2 2 2 3 2

0 0 1 0 0 0 1 2

0 0 2 3 1 0 2 0

0 0 3 3 2 0 3 3

a

Fluorescence was coded as follows: ‘0’ fluorescence not higher than in cells of the healthy section of the same tissue; ‘1’ weak signal (fluorescence restricted to a few cells); ‘2’ intermediate signal (fluorescence in half or almost half of the cells); ‘3’ strong signal (fluorescence in all the cells). b In situ hybridisation was coded as follows: ‘0’ signal not higher than in cells of the healthy section of the same tissue; ‘1’ weak signal (purple coloration restricted to a few cells); ‘2’ intermediate signal (purple colouration in half or almost half the cells); ‘3’ strong signal (purple colouration in all the cells).

1 week later (Fig. 4). Taking together, the mestome and the vascular bundle sheath were the most discriminant tissues for resistance among dates and among lines, RNA and CP being detected 14 DPI in IR64, but only 1 week later, at 21 DPI, in the NIL and in Azucena (Table 2). The length and the timing of the delay in virus detection in the NIL at tissue levels in the mestome and in the vascular bundle sheaths paralleled those found at leaf and plant levels through symptom expression and virus accumulation.

4. Discussion There is little information on the genetic basis of tolerance to plant viruses [19]. Tolerance is sometimes considered as an eroded form of resistance, whereas response to Lettuce mosaic virus (Potyviridae) involved genes which were either resistant or tolerant genes depending on the viral isolate they encounter [14]. By contrast, tolerance to RYMV, expressed by mild symptoms on leaves and a limited impact of growth despite

the generalised distribution and high accumulation of the virus, had a specific genetic determinant. Tolerance and resistance to Tomato yellow leaf curl virus (Geminiviridae) were found in different cultivars [33], whereas for RYMV they were associated within the same cultivar Azucena, and probably both present in most upland japonica varieties [6]. Among the six genomic regions associated with the DH response to infection, some QTLs are possibly involved in tolerance. In particular, QTL1 was associated with a limited impact of infection on plant growth and yield, and with the presence of the Azucena allele of the sd1 gene on chromosome 1 [6,7]. However, tolerance was found recently in an improved japonica cultivar with indica-like growth characteristics (our unpublished data). Similarly, the major QTL for tolerance to Barley yellow dwarf virus (Luteoviridae) in oat was not associated with morphological traits [21]. Differences in symptom expression between tolerant and susceptible rice lines may reflect, alternatively, a different cell localisation of the virus, possibly in the vacuoles, as suggested in Ref. [13]. Partial resistance was dissociated from tolerance and other morphological and physiological Azucena traits by

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Fig. 4. RNA localisation by in situ hybridisation (purple spots) in semi-thin sections of systemically infected leaves of the Oryza sativa indica cultivar IR64 (top), of the IR64-nearly isogenic line (NIL) carrying the major QTL (QTL12) of the partial resistance introgressed from the japonica cultivar Azucena (middle), and Azucena (bottom), 14 (left) and 21 days post-inoculation (DPI) (right) in leaf tissues: bundle sheath (bs), higher epidermis (he), lower epidermis (le), mesophyll (mp), mestome sheath (ms), phloem parenchyma (pp), sclerenchyma (sc), sieve tubes (st), xylem vessels (xv) and xylem parenchyma (xp).

the NIL approach. The NIL provided a good confirmation of the effect of QTL12 through the (IR64 £ Azucena) DH population analysis, and indicated that the QTL was expressed similarly at plant, leaf and tissue levels in a sativa and in a japonica genetic background. The lower level of resistance sometimes observed in the NIL likely reflects that other QTLs were also involved in partial resistance. Although several co-localised with QTLs involved with morphology and development in Azucena [7], response of the NIL with an indica background confirmed that no morphological or physiological traits of Azucena were involved in the partial resistance associated to QTL12. Thus, partial resistance was apparent at an early stage of infection and characterised by a 1-week delay in virus accumulation and tissue invasion. Passed this delay, high virus content, generalised tissue invasion and a pronounced necrosis developed, showing that partial resistance was transitory. In tissues, this was observed in the NIL with the CP and RNA detection which targeted the virus either as

virions or as a nucleo-protein complex [11,12,28]. Delayed detection of RYMV did not result from the overall slower metabolism of japonica rices as hypothesised earlier [25], as the same time lag was observed in the NIL with an indica background. The delayed virus detection and symptom expression at plant, leaf and tissue levels suggests that partial resistance is due to an impaired virus movement. The normal virus replication in protoplasts of resistant cultivars supports the hypothesis of resistance via restricted virus movement [25]. Transient partial resistance, due to delayed virus movement, may be a general feature of such resistances since, once the tissues are invaded, the plant responds as a susceptible host. The interaction between viral and host proteins are necessary for virus movement. The recessive genetic determinism of the partial resistance to RYMV [4] suggests the absence or mutation of a host component necessary for virus transport coded by the Azucena allele of the QTL12. Partial resistance was associated consistently with a delayed invasion of the mestome and of the vascular bundle

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sheath. Actually, vascular and mesophyll tissues differ functionally, the bundle sheath – phloem interface is critical for virus trafficking into, through and from vascular tissues [26] and can be an efficient barrier to virus movement [32]. The appearance of dot symptoms was the first manifestation of RYMV infection. These dots may be discrete foci that occurred where the virus left the vascular tissues after passing through the mestome, and the associated streaks may result from cell-to-cell diffusion in the mesophyll from these unloading points. Then, partial resistance may be due to impaired virus movement through the mestome, and subsequent restriction of the number of initial mesophyll foci. This hypothesis is being tested through cytological investigations of the mestome and of the neighbouring dots after inoculation with GFP-marked isolates. Transfer of partial resistance of the upland japonica rice varieties into the susceptible lowland varieties by backcross breeding was successful. QTL12 and QTL7 conferred a 1-week delay in infection by the reference isolate, representative of the pathogenicity of most RYMV isolates. However, although partial resistance is a useful feature, it not sufficient alone to restrict virus diffusion and multiplication in indica lines, and should be combined with other genes of resistance. Introgression of the additional 15 resistance QTLs is not feasible and unlikely to substantially improve the outcome of the response of the introgressed lines. Tolerance is also not appropriate to control RYMV. First, severe isolates found in the fields or generated by serial inoculation can overcome tolerance and resistance in Azucena [16]. Second, tolerant lines will provide a potent virus reservoir and lead to spread to neighbouring susceptible rice. Pyramiding different sources of resistance has been recommended to increase the resistance durability [18]. Interestingly, isolates able to overcome tolerance in Azucena did not break the high resistance of Gigante and vice versa [16]. Therefore, in addition to seeking other sources of resistance, it may be advantageous to combine different sources of resistances to RYMV.

Acknowledgements The first author was supported by a grant from the State Scholarships Foundation of the Republic of Greece. We thank J. M. Thresh for helpful criticisms of the manuscripts and J. Aribi for technical assistance.

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