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u gq O 1999 Kluwer Academic Publishers. Printed in the Netherlands. Molecular Biology Reports 5: 95-102, 1999. 95

Resistance to African cassava mosaic virus conferred by a mutant of the putative NTP-binding domain of the Rep gene (AC1) in Nicotiana benthamiana

Abdourahamane Sangaé1, Dalun Deng2, Claude M/bauquet2** and Roger N. Beachy2 lhboratoire de Gkzétique de I’UFR des Biosciences, Urtiversite‘ de Cocody, 22 BP 582, Abidjan 22 Ivory Coast; ’ILTABflSRI, Division of Plarzt Biology, BCC 206, 10550 North Torrey Pines Road, La Jolla, CA 92037, USA (*autlzor for correspondence; E-mail: iltab @scripps.edu) ‘

Received 14 July 1998; accepted in revised form 15 September 1998

Key words: AC1 mutant, ACMV, pathogen-derived resistance, NTP-binding site

i Abstract ’

We construdted a mutation in DNA A of African cassava mosaic virus (ACMV) to alter the putative NTP-binding site in the replication-associated protein gene (AC1). When transgenic Nicotiana benthamiana plants expressing the mutated AC1 gene were infected with A C W , the plants exhibited tolerance to infection consisting in a delay in symptom appearance and/or the presence of mild symptoms. In addition, the resistant plants accumulated less viral DNA than non-transgenic plants. As judged by northern blot analysis and symptom development of segregating progeny from different lines, a high level of expression of the mutated AC1 gene is essential for the development of resistance. Issues related to the use of different versions of AC1 for the control of ACMV are discussed.

Introduction

Cassava is a major food crop in Sub-Saharan Africa and is cultivated for its starchy roots and protein-rich leaves. The most economically important disease af- fecting cassava in Africa is cassava mosaic disease (CMD) caused by African cassava mosaic geminivirus (ACMV) [3, 121. Newly emerging strains and/or species of geminiviruses related to ACMV 1211 often cause severe variants of the disease and have been re- ported to be a real threat to food safety on the African continent [29]. Resistant varieties produced through classical breeding [14] are being overcome by these viral strains or species.

Begomovirus [30, 331. Component A of the genome encodes on the virion strand, the coat protein (AVl) gene [37], and there are three overlapping genes on the complementary strand. Of the latter, ACl, the replication-associated protein gene (or Rep gene), is indispensable for the replication of both genomic com- ponents [ll]; AC2 is thought to be involved in the trans-activation of sense gene transcription from both

J

ACMV is a two-component whitefly-transmitted .

A and B components [ 111; and AC3 is implicated in ef- ficient replication of the virus [ 1 11. The B component encodes one gene in the sense-orientation (BVl) and

. anot$e$ o k j n the complementary,s”eqse (BCl), both of which are essential for virus movement [lo, 361.

Information about the putative functions of gemi- nivirus genes have led to the development of virus con- trol strategies based on the expression of virus-derived genes in transgenic plants. Coat protein-mediated re- sistance [26] as well as anti-sense RNA sequences derived from AC1 [2] have been reported to con- fer limited resistance to the Israeli isolate of tomato yellow leaf curl (TYLCV-Is). Antisense-based strate- gies were similarly found to confer resistance against tomato golden mosaic virus (TGMV) and beet curly top virus (BCTV) in tomato [l, 61. Other strategies, such as the use of defective-interfering viral DNA [32] or the expression of dianthin, a ribosome-inactivating protein [19], were also reported to confer resistance to ACMV. More recently, Hong and Stanley [20] re- ported that virus resistance was conferred in Nicotiana benthamiana by a full-length gene of ACMV AC1.

V

J 96

1 5 3 4 6 f

BclI (1286) * -- + . sPy(2581) , f 4 f , -

AC3

n i o ~ & V A E T m Defective mutant (mlAC1)

Figure 1. Mutation in the putative NTP-binding domain of AC1 gene. The region of ACMV DNA-A subjected to mutations is shown as a discontinuous line. Nunibers on top of short arrows (1 to 6) cor- respond respectively, to oligonucleotides ac-I to ac-6 described in Table 1. Position of SphI and BclI sites of ACMV DNA-A are indi- cated between parentheses. Long arrows represent coding sequences of ACl, AC2 and AC3 genes as indicated. The relative scale is not respected as shown by double dashes (II). The white box below the coding sequences represent the AC1 protein. Hatched box indicates the putative helicase domain and black boxes, the conserved motifs A, B and C as shown. The amino acids that have been changed h the box A of the putative helicase domain are also shown.

This group also showed that AC1 containing a stop codon after 57 N-terminal amino acids, and a fusion protein comprising those 57-N terminal amino acids of AC1 and 93 amino acids unrelated to AC1, also interfere with viral DNA replication in N. tabacum protoplasts. Noms et al. [28] also obtained transgenic N. bentlzamiaiza lines resistant to TYLCV using a con- struct encoding the N-terminal 210 amino acids of TYLCV C1 (AC1 homologue). In this work, we report the use of a replication-defective mutant of ACMV AC1 that is altered in its putative NTP-binding do- main [13] to control ACMV infection in transgenic N. benthamiana.

Materials and methods

Reagents, enzymes and ACMV clones

Restriction and modifying enzymes as well as syn- thetic oligonucleotides were obtained from Gibco- BRL (Bethesda, MD). The polymerase chain reac- tion (PCR) was performed with a kit from"Perkin Elmer Cetus (Foster City, CA) according to the manufacturer's recommendations. The plant vector pMON977 which contains an expression cassette of the cauliflower mosaic virus (CaMV) enhanced 35s promoter and the pea Rubisco E9 terminator was ob- tained from Monsanto (Saint Louis, MO). Infectious ACMV DNA clones (pCLV1.3A and pCLV2) [24] were kindly provided by Dr John Stanley (John Innes . Institute, UK).

Construction of AC1 mutants

The AC1 gene was mutated in the putative NTP- binding domain (E21gGDSRTGKT227) using site- directed mutagenesis by overlap extension [ 171 with a set of four synthetic oligonucleotides: acl-I, acl- 2, acl-3 and acl-4 (Table 1). Using pCLV1.3A as DNA template, acl-1 and acl-2 were used to am- plify one half of AC1 gene. Similarly, the other half was amplified with oligonucleotides acl-3 and acl- 4. The amplified products were subsequently fused by PCR using acl-1 and acl-4 as primers. The resulting full-length mutant was cloned into the BamHI-$acI sites of pMON 977 to construct pILTAB194. The nu- cleotide sequence of the insert was confirmed before transferring the construct to Agrobacteriunz.

In order to test the effect of these mutations on virus infectivity, ACMV DNA component A was mod- ified to contain the mutant mlACl using site-directed mutagenesis by overlap extension with acl-3 and acl- 4 as mutating oligonucleotides and acl-5 (upstream) and acl-6 (downstream) as cloning primers (Table 1). The mutated gene was cloned in place of the BcZI (1286)-SphI (2581) fragment of pCLV1.3A [33]. The mutation was confirmed by DNA sequencing of a selected clone and is designated pCLVl.SA/ACl-.

Transformation of N. benthamiana plants

Plasmid plLTAB194 was mobilized into A. tumefa- ciens strain ABI carrying the disarmed Ti plasmid pMP90RK 12.51 using the triparental mating technique with the helper plasmid pRK2013 [8]. Transformation of N. benthainiana leaf disks and regeneration of trans- genic plants were carried out as previously described [22,23].

Nucleic acid analysis

Total DNA was extracted from N. benthanziaiia plants as previously described [7]. RNA extraction as well as Southern and northern blotting hybridization re- actions were performed according to standard proce- dures [31]. DNA labeling was performed with the random priming 'Prime It-II' kit (Stratagene, San Diego, CA) according to the manufacturer's protocols.

Karianiycin selection and infection of transgenic plants

Transgenic seeds were grown on MS medium solid- ified with agar I271 and supplemented with 20 gll

, .. .. . . , . , . . ....I_ ...__ ~ . ../,_._ ...... "__. ..._ ̂ . . , I . .

. , /

-.

Table I. Sequences of oligonucleotides used for mutation and cloning purposes.

.. , .._.I- .-

97

Designation Corresponding sequences

ad-1 5'-G CAGGCCTGAGCTC ATGAGAACTCCTCG'MTI'CGAA'IT-3' ad-2 ad-3 ad-4 5'-CGGGATCCCTACGCCGGATGGCTC-3'

5'-TATCGTCTTCGCTAC TA TGGC AG AAGCTTCTATGAC-3' 5'-GTCATAGAAGC l T C TGC CAT AGT AGC GAAGACGATA-3'

ad-5 5'-CAAAACATGATCAGCAGCTC-3' ad-6 5'-CTCACTTGCATGCCCTC3'

acl-I contains the translation start codon (bold characters) and the first 21 nucleotides of the AC1 coding region. Nucleotides corresponding to the Sai1 and Sac1 sites (underlined) were added to the 5' end of the oligonucleotide for cloning purposes; ad-2 contains ACMV DNA-A sequences from nucleotide 2073 to 2108, except for intentionally substi- tuted nucleotides (in bold italics); ad-3 is complementary to ad-2; ac14 contains the complementary sequence of the AC1 gene translation termination codon (in bold) and the 13 upstream nucleotides as well as a BamHI site at the 5' end (underlined); acl-5 corresponds to nucleotides 1286 to 1305 of ACMV DNA A and contains a BclI restriction site (underlined) whereas ncl-6 is complementary to ACMV DNA A nucleotides 2581 to 2787 and contains an SphI site (underlined).

sucrose and 150 mgA kanamycin. After two to three weeks of growth, plantlets were transferred to soil for one week and regularly misted with water. They were then transferred into individual pots with fertil- ized soil, grown for an additional week and infected with ACMV.

Genotypes of individual R1 plants were deter- mined using a kanamycin test as follows: leaf disks (sterilized in 20% bleach, 1% wlv SDS for 7 min) were collected from transgenic plants three days before in- fection and placed on MS medium containing 20 gA sucrose, 2 mgA 1-naphthaleneacetic acid and 100 mgA of kanamycin. After two weeks of growth, kanamycin resistance growth could be clearly identified: tissues from plants that did not express nptII turned yellow andor brown as they died while kanamycin-resistant tissues remaineld green. In some experiments, geno- types of individual plants were determined by PCR amplification with a d - l and a d - 4 as primers.

Mechanical infection of Nicotiana benthamiana

N. benthaminna plants 4-5 weeks old (having four 'fo five leaves) were mechanically infected with crude sap extracted from young leaves of infected plants showing typical ACMV symptoms. The infectious sap was diluted (1 g leaves in 100 ml 0.1 M potas- sium phosphate, pH 7) and rubbed onto the surface of the youngest fully expanded leaves (usually the second and the third leaves from the apex) that were previously dusted with carborundum. In the case of infection by claned DNA, 0.5 &g (in 0.1 M potas-

sium phosphate, pH 7) each of DNA A (pCLV1.3A or pCLV1.3A/ACl-) and DNA B (pCLV2B) was used.

Scoring disease symptoms

Symptoms caused by ACMV were recorded daily from the date of inoculation and plants were scored as follow: O = no symptoms; 1 = local chlorotic spots and/or mild leaf curling of young systemic leaves; 2 = severe leaf curling, only on young systemic leaves; 3 = curling of various systemic leaves (different level of severity could be observed); 4 = large chlorotic spots on infected and systemic leaves, curling is severe in all symptomatic leaves; 5 = typical mosaic symptom.

Analysis of an AC1 mutant

The ACMV Rep gene was mutated in the re- gion coding for the putative NTP binding domain (E219GDSRTGKT227) by site-directed mutagenesis (Figure 1). In order to avoid reversion of the mu- tation by a single-nucleotide change, several amino acids were modified. These changes were performed according to Taylor [34] in such a way that putative conformational changes that might occur at the protein level are minimized. The mutated version of the AC1 gene, which contains seven altered amino acids, has the sequence E21gASALKET227 and will be referred to as mlAC1.

. .

c

Bl 98

Table 2. Inoculation of N. beiirhaniiana with cloned ACMV DNA

Experiments Inoculuma pCLV 1.3NACI- pCLVl.3A pCLV1.3A +pCLV 2B +pCLV1.3NACI- +pCLV2B

SpCLV2B

1 Ob/] o 919 Ill1 1 2 0/13 10110 10110 3 0110 15/15 10110

"Nbeiitlzaminna plants were mechanically inoculated with 0.5 p g of each DNA A and DNA B. bNumber of symptom-bearing plants over total inoculated plants.

In order to characterize the biological activity of the NTP-binding mutant, we constructed a mutant of ACMV DNA A containing. mlACI. The mu- tant DNA A (pCLV1.3A/ACl-) was co-inoculated to N. benthamiana with wild-type DNA B (pCLV2B) alone, or together with wild-type DNA A. In sev- eral experiments, pCLVl.3NACl- inoculated with pCLV2B was found to be non-infectious under condi- tions in which infection by the wild-type (pCLV1.3A + pCLV2B) infected all the N. benthamiana plants inoculated (Table 2). When pCLV1.3NACl- was Co-inoculated with both pCLV1.3A and pCLV2B, all challenged plants were infected 20 days after inoc- ulation (Table 2). No significant delay in symptom development was observed between N. bentlzarniana plants inoculated with wild-type DNA and those chal- lenged with a mixture of DNA that contained that defective DNA A.

Characterization of transgenic plants

Eleven independent kanamycin-resistant plants were obtained from N. bentharniana transformed with pILTAB 194, seven of which were found to contain the complete inlACl as judged by PCR amplifica- tion (Table 3). Transcripts of the gene were detected in all those seven lines although considerable vari- ation in the relative transcript levels was observed (Figure 2). These transgenic lines were fertile and, based on the kanamycin greening assay performed with the progeny of each individual RO plant, only line B contained more than one transgene locus (Table 3).

Transgenic rizlACl plants are resistant to ACMV

bzfection of karzainyciiz-resistant RI m l AC1 plants In order to evaluate the biological effect of the AC1 gene in transgenic plants, seeds collected from RO

A B C F G J K 977-1

1.25 k b -b

Figure 2. Northern blot of mlACl transgenic RO plants. Total RNA (20 pg per line) extracted from transgenic mlACl lanes (A to K) and from transgenic plant containing the vector only (977-0 were hybridized with the mlACl coding sequence labelled with ( Y - ~ ~ P .

80 t

5 10 i 5 20 25

t - m l A C 1 - K

Days anfr inoculation

Figure 3. Symptom development in mlACl transgenic Nico- tiaiza beiztlzariziana plants selected on MS medium supplemented with kanamycin at 150 mg/l. mlACI-A to K transgenic lines con- taining mlACl gene; 977-C a transgenic line containing the vector done.

plants (regenerated plants) expressing AC1 were se- lected. As shown previously (Table 3), all of the seven transgenic lines expressing the AC1 gene also con- tain the nptlI gene so the AC1 encoding plants were indirectly selected on kanamycin-containing medium. These plants were then challenged with ACMV. Fig- ure 3 summarizes the results of infection experiments carried out with at least 10 kanamycin-resistant plants iiom the R1 generation per line. When selected on medium containing a high concentration of kanamycin (150 mg/l), the proportion of infected plants 25 days after ACMV inoculation ranged from 25 to 40% while 100% of untransformed N. benthamiana plants and 95% of plants transformed with the vector alone (pMON977) became infected. In addition, ca. 50% of the plants that became infected showed relatively milder symptoms (ranging from level 2 to 3) during the early stages of the infection. The majority of con- trol plants displayed severe symptoms as compared to iitlACl transgenic plants in all experiments.

Analysis of three segregating lines R1 plants grown without selection on kanamycin were produced from lines A, E and C and at least 45 in-

. .

i Table 3. Characterization of mIACl transgenic plants.

Lines Gene integrity” Expressionb mlACI Number of challenged Number of challenged RI d A C l nptII locus #‘ KanR plantsd segregating plantse

A + + 5+ 1 45 119 B - I - + + >2 26 n.d. c + + + 1 30 100 D P + - n.d. n.d. n.d. E f + + 1 n.d. n.d. G + + 5+ 1 20 n.d. H P + - n.d. n.d. n.d. I P + - ad . n.d. n.d. J t + 4+ 1 25 45 K + + 2+ 1 20 n.d.

n.d.: Not determined. “Integrity of transgenes was analysed by PCR amplification of cassettes containing either mIACl (driven by enhanced 35s promoter and E9 terminator) or npfZI (driven by regular 35s promoter and Nos terminator). + complete cassette amplified; p: cassette found partial after amplification. Expression level was judged by Northern blot analysis (Figure 2). The number of + arbitrarily designates the =Iative expression of each line as compared to the RNA accumulation in h e B; - indicates no detectable RNA. ‘Transgene loci number was estimated by kanamycin resistance test. dR1 plants were selected on medium containing either 100 mgfl or 150 mg/l of kanamycin (see text). ‘RI segregating plants tested for ACMV resistance.

dividuals from each line were randomly chosen for infection experiments. The genotype of each indi- vidual was determined by PCR and symptom devel- opment was scored. PCR-negative plants (segregants lacking the mlACl gene) and pMON977 transgenic plants were used as controls. As expected (based on to transcript levels), the three lines showed different behavior with respect to symptom development. In lines E, and C, no significant differences in symptom severity was observed between mIAC1 PCR-positive plants and control plants. On the contrary? line A was found to be resistant to ACMV infection, i.e. fewer individuals in that line became infected and those in- fected developed mild symptoms compared with the vector-only plants. A perceptible delay in symptom development was noticed between the mlACl PCR- positive plants and the two sets of controls (Figure 4). No significant differences were observed among the two groups of controls (i.e.? mlACl(-) and 977-C lines) where overall infection was ca. 90%.

Fstribution of symptom severity during infection As indicated earlier, the ACMV symptoms in R1 trans- genic plants selected on kanamycin were milder than typical symptoms caused on wild-type N. benthami- ana. In order to better describe this phenomenon, heterozygous offspring of two high expressor lines (A and J) were infected with ACMV and the symp- toms were scored daily. Vector-only transgenic plants and offspring of line A lacking the mlAC1 gene were

99

700 7

O i I 5 10. 15 20 25 30

Days after inoculation

-a- ml AC1 -

Figure 4. Bo+ieg)egãtion of ACMV resistance phenotype and mlACl gene in an R1 population of transgenic Nicotiana ben- thainiana plants from line A. mlACI+: segregants from plant line A containing mlACl gene; mlAC1-: segregants lacking mlACl gene; 97742 a transgenic line containing the vector only. I

used as controls. The distribution of different levels of symptom seventy after infection with ACMV is shown in Figure 5. A clear difference is observed between segregating transgenic plants that contain the mlACl gene and control lines. Overall, control plants ranged from 90 to 95% of symptoms classified as level ‘5’ 15 days after infection whereas, in line A for example, 20% were scored as 5, 13% as 4 and the remkining 67% ranged from score of ‘1’ (17%) to ‘3’ (23%) . At the same time point, 10% of the inoculated plants of

1 O0

line 5 remained symptomless and 23% were scored as ‘1’. The proportion of plants scoring between 2 and 5 ranged from 5% to 20% for that line. Viral DNA accumulation in the transgenic plants was found to coirelate with symptom severity (Figure 6 ) suggest- ing that the resistance is linked to inhibition of viral DNA replication. However, further investigations are needed, particularly in protoplasts, to assess the actual effect of mlACl on viral DNA replication.

Discussion

We constructed a version of a cloned ACMV DNA A carrying a mutation in AC1, the replication associated protein, altering its putative NTP-binding site. The mutations modified amino acids of the putative motif A, including the highly conserved lysine residue at po- sition 226 [13]. DNA A that was mutated in this motif was not infectious when inoculated to N. beitthamiaizn with the ACMV DNA B clone. Even though we did not assay the mutant protein for NTP-binding capac- ity, the loss of activity confirms the importance of the mutated amino acids in the function of the replication associated protein [ 161.

When transgenic plants expressing the mutated AC1 gene were infected with ACMV, we observed a delay in symptom appearance andor the presence of mild symptoms. In addition, the resistant plants accu- mulated less viral DNA than plants that did not express the transgene. These results suggest that constitutive expression of the mutant gene affects viral DNA repli- cation, but additional work is needed in protoplasts to confirm that effect.

In addition, northern blot analyses and studies on the development of disease symptoms in segregat- ing progenies of different plant lines indicated that a threshold level of expression of the mutated AC1 gene is essential for the development of resistance. When selected on high concentration of kanamycin, only homozygous plants and lor particular individu- als expressing a high level of transgenes are selected. Therefore all lines containing mlACl gene behave similarly as the selection picks up those genotypes. However, in natural conditions, only lines that have a constitutively high level of expression of the trans- genes i.e. A, G and J (attested by the northern blot pattern) develop resistance.

Strategies for engineering virus resistance based on virus replication-associated proteins are usually designed either to inhibit production of the protein

by disrupting the transcription of the gene andlor the stability of the transcript, or to produce a mu- tant that restricts its function. Expression of wild-type replication-associated protein genes as well as mutants of these genes in transgenic plants has been reported to interfere with replication of RNA viruses [4, 5, 91. In the case of geminiviruses, Hong and Stanley [20], demonstrated, that expressing full length ACMV AC1 in transgenic N . benthainiana produced plants that were resistant to virus infection. Moreover, ex- pression of AC1 under the control of the CaMV 35s promoter in N. tabncuin BY-2 protoplasts was found to reduce the level of replication of infectious DNA A [20]. Expression of a truncated TYLCV Rep gene in transgenic plants gave comparable results [28].

In addition to its involvement in replication, the AC1 protein is known to negatively regulate its own gene expression [18, 201 probably through competi- tion with transcription factors. The N-terminus of the AC1 protein appears to be involved in this interaction because in protoplasts it was demonstrated that a very short segment of AC1 (the 57 N-terminal amino acids) is sufficient to inhibit ACMV DNA replication [18, 201. Thus, it appears that all the AC1 constructs tested, including the one reported here, confer similar type of resistance. We did not analyze the accumulation of mlACl protein in this study but we propose that the constitutive expression of the mutant protein interferes with the virus replication by interacting with regula- tory sequences located upstream of the AC1 gene as suggested by Noms et al. [28].

In the case of the full-length ACMV AC1 gene, even though that construct was not proven to be functional in transgenic N. beizthainiaaa, one can- not rule out the possibility that the unmutated AC1 could well be functional for example in cassava as TGMV AC1 was found to be in tobacco [15]. Thus, attempting to control ACMV infection by expressing the wild-type AC1 gene carries certain risks particu- larly if the gene was to be expressed in cassava. It should be mentioned, referring to the case of TGMV, that no resistance was observed when cloned wild- type TGMV DNA was agro-inoculated in transgenic tobacco expressing a functional Rep gene.

These analyses led us to the construction of a mu- tant that is altered in a domain that is known to be indispensable for normal function of the protein. We have intentionally targeted many sites in order to avoid a reversion of the mutant. However, we did not alter

i I I

i i other important domains of the putative helicase for they may contain at least part of the single-stranded

i

I

I 1

I I

-.

---T77- . . I .-.....-I.__ l-__._l_.._.?.. ~.,~ .,_.__._- . . .. .. i - ;.*,.

. . . .

101

977-163

35 I

o 5 10 15 20

Days after inoculation I

mlAC1-27

E Days after inoculation

m l AC1 -A8

35 I

O 5 10 15 Days after inoculation

20

I

I m l AC1-J6

30 I

O 5 10 15 20

Days after inoculation I I

Figure 5. ACMV symptom severity in transgenic lines expressing or not the mlACl gene. O to 5: ACMV symptom scores as described in Materials and methods. mlAC1-27 and mlAC1-J6 heterozygous descendants of line A and J respectively, segregating for AC1 gene; mlAC1-AS: homozygous descendant of line A lacking the mlACl gene; 977-163: heterozygous descendants of line 977-1 segregating for the vector only.

b b b a b b b a a c b b a b a a b c c c h c h a h h b b b c

3 3 3 2 5 4 5 1 1 3 2 1 2 2 1 2 4 5 5 4 2 5 2 2 3 . 3 4 1 3 5 +. e ' ' Y I

Figure 6. Accumulation of viral DNA in 30 inoculated offspring of mlACl 'line A; 1; days after infection. Total DNA extracted from the two first apical leaves were subjected to electrophoresis in 1% agarose gel and transferred DNA was hybridized with ACMV coat protein gene probe. The number under each track indicates the symptom score for the corresponding plant. Genotype of each plant was determined by kanamycin test carried out with seeds collected from each individual plant and is shown above the tracks as it follows: 'a' = mlACl homozygous plah&; 'b' = ntIACI heterozygous plants; 'c' = offspring without mlACl transgene.

DNA binding property. Our primary goal was to test whether such a mutant could give comparable results to wild-type and truncated versions of the Rep gene. Our report shows that tolerance to ACMV infection can also be achieved with AC1 mutants and that vir- tually symptomless plants can be obtained, provided that the mutated gene expression is sufficient.

The main host of ACMY being cassava, our efforts are oriented toward testing different defective AC1

mutants that we believe are safer to use în transgenic cassava.

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219-225 (1996).

,2443 (1993).

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204 383-396 (1986).

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New Strategies in Plant Improvement

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Editor-in-Chief

l i Joseph N.M. MOL, Amsterdam, The Netherlands ! %

Editorial Board

Pam e I a DU N S M U I RI Oakland, California , U. S. A. Robert M. GOODMAN, Madison, Wisconsin, U.S.A. John A. HOWARD, Johnston, lowa, U.S.A. Horst LÖRZ, Hamburg, Germany Marnix PEFEROEN, Ghenf, Belgium John RYALS, Cay, North Carolina, U.S.A.

Francesco SALAMINI, Köln, Germany Takuji SASAKI, Ibaraki, Japan Wolfgang SCHUCH, Bracknell, United Kingdom Toni SLABAS, Durham, United Kingdom Pierre J.G.M. DE WIT, Wageningen, The Netherlands Nevin D. YOUNG, St. faul, Minnesota, U.S.A.

I

" Y M.A. SAGHAI MAROOF, Blackiburg,' tir&&, U.S.A.

International Society for Plant Molecular Biology

Volume 5, No. 2, 1999

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ISSN 1380-3743 CODEN MOBRFL

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