Potato virus Y

FIG. 2. PCR assays of transgenic plants.

(A) Alignment of primers with the inserted PVY1.4NIa. Primer 1 aligns with the promoter that is not transcribed to RNA. Primer sequences are given in the text. (B) Sample of a Southern blot of the PCR product of transgenic (V2, V3) plants and of a nontransformed (SR1) plant.

Lanes 1 and 2, DNA-PCR of V2 using primers 1 (specific for DNA only) and 11 or primers 10 and 11, respectively; lanes 3 and 4, RNA-PCR of V2 using primers 10 and 11 or primers 1 and 11, respectively; lane 5, stained size markers (not seen in the autoradiogram); lanes 6 and 7 and 8 and 9, RNA-PCR as in lanes 3 and 4 but with V3 or the nontransformed SR1 plants, respectively. Arrowheads indicate positions of the respective bands.

NPTII-homozygous R2 plants derived from the resistant individuals of lines V2 and V3, as well as from two PVYsusceptible lines, were selected for further analysis. All descendents of the resistant plants remained resistant, and those of the susceptible plants remained susceptible. The presence of PVY1.4NIa in these R2 plants was assayed by PCR using primers 1 and 11 (Fig. 2). Expression of NIa was tested by NA-PCR using primers 10 and 11 (Fig. 2A). As a safeguard against possible DNA contamination, a second RNA-PCR, employing primers 1 and 11, was carried out in each case. Since primer 1 represents promoter sequences, any positive PCR band appearing under these conditions would indicate DNA contamination. Each ofthe transformed R2 plants, whether PVY resistant (Fig. 2) or susceptible (data not shown), expressed the NIa cistron. The various stages of selection of NIa-resistant plants, the types of analysis at every stage, and a short account of the results are summarized in Table 1. Expression and processing of pG3PVY3.9-transformed plants are referred to below. A ing Protection from PVY Infection. The following tobacco plants were assayed for viral resistance: nontransformed SR1, transformed with an "empty assette" (insertless pGA643), R1 transgenic NIa+, and the above-described R1 NIa- plants. Plants were inoculated with PVY and inspected daily for symptom appearance. Viral accumulation was determined by ELISA at 4- to 5-day intervals. The R1 progeny of transgenic lines V2 and V3, each carrying two copies of the NPTII gene, and the progeny of line V6, carrying a single copy of that gene, were selected for protection studies. As shown in Table 2, all descendants of V2 and V3 exhibited either partial resistance to viral infection, in which symptom appearance was delayed, or "complete" resistance, where symptoms did not appear at all during the 50-day experimental period (Fig. 3). No viral accumulation could be detected in the resistant plants (Fig. 4A). Similar experiments were performed with transgenic plants carrying the NIa-Nlb-CP (pG3PVY3.9) construct. Members of two R1 lines (110 and I33) were found to be resistant to PVY according to the criteria used for NIa-transformed plants (an example is shown in Figs. 3 and 4B). However, in this case, the element responsible for resistance is uncertain, since these plants also carried the CP cistron. The only known tobacco-infecting potyvirus in Israel is PVY. Hence it was not possible to check resistance of the reported transgenic plants to other potyviruses. However, resistance to the unrelated tobacco mosaic virus (TMV) was tested. Three PVY-resistant V3 (pGNIal.4+) and three PVYresistant 110 pG3PVY3.9+) plants were inoculated with 20 ,ug of TMV per ml. Visual observation did not indicate any resistance to TMV, as the severity of symptoms and the time of symptom appearance were similar in the control and the PVY-resistant plants. Ten resistant V3 and I10 plants, as well as control, nonresistant plants, were kept at 35C for 48 hr and then inoculated with PVY and the temperature was shifted back to 260 C. In this case, very mild symptoms did develop (compared to severe symptoms in the control plants), with the virus titer in the V3 and I10 plants being <40% of that in the controls (Fig. 4C).

FIG. 3. Demonstration of resistance to PVY.

The plants were inoculated with 30 ,g of PVY per ml. (A) Resistant plant (line V2) transformed with pGNIA1.4 carrying the cistron for a PVY protease (NIa). (B) Control plant transformed with the insertless vector pGA643 (Km+; NIa). (C) PVY-susceptible V2 plant that has lost the inserted sequences upon segregation (Km-; NIal). (D) Resistant plant (line 133) transformed with pG3PVY3.9 carrying the NIa-NIb-CP cistrons.

DISCUSSION

Plants transformed with viral structural gene sequences have been shown to acquire resistance to infection by the same, or related, viruses (reviewed in ref. 22). Resistance has also been reported in tobacco transformed with a nonstructural viral sequence, the putative TMV replicase (23). The present paper reports resistance in plants transformed with yet another nonstructural gene, coding for a protein with a different viral function (a protease). The original transformed plants were propagated by self-pollination for two generations, and all descendants of resistant R2 plants maintained their resistance. The copy number of the inserted NPIII gene could be determined from the R1 segregation pattern for Km resistance, and NPTII homozygosity could be determined from R2 Km+ segregation. Due to linkage, these parameters are also indicative (although not necessarily conclusively) of NIa homozygosity, since many cases of sequence "escapes" have been recorded (23). However, in the presently reported case, the NIa-NPTII linkage was preserved, as the R2, NPTII-homozygous progenies did not segregate when tested for PVY resistance. As an arbitrary starting point at the R1 stage, the two transgenic lines (V2 and V3), presumed to carry two copies of the NIa gene, were included in the first sample taken for a study of resistance. As indicated in Table 2, all 10 R1 progeny in these lines exhibited some degree of resistance, ranging from delayed symptom appearance (and much milder symptoms) to total resistance throughout the 50-day experimental period (in about 50% of the cases). On the other hand, line V6, carrying a single copy of the insert, exhibited only a short delay in symptom appearance and could not be considered resistant. However, the sample sizes were too small to validate the necessity of a higher copy number to obtain the observed resistance. Further conventional analyses, by crossing the hundreds of yet untested NIa+ transformed plants, are required before any conclusion can be drawn. Vpg, the protein that is covalently bound to the 5' end of potyvirus RNA, is thought to play a role in viral RNA replication (24-26). It has been recently suggested that the N-terminal portion of NIa is, in fact, a part of Vpg (26, 27). Hence, the cloned NIa, being defective at its N terminus, may be inactive with respect to RNA replication. The binding ofthe inactive product of the cloned PVY1.4NIa to the 5' end of the viral RNA may block a fundamental step in viral RNA replication, thereby protecting the plant.

FIG. 4. PVY accumulation in tobacco leaves as determined by ELISA.

(A) pGNIal.4-transformed plants. The control curve represents data obtained from plants transformed with the insertless vector pGA643. Control curves obtained from nontransformed plants and NIa- plants were similar. V2 and V3 represent PVY accumulation in four orfive R1 transformed, fully resistant plants. (B) pG3PVY3.9-transformed plants. 110 and I33 represent average values in four R1 resistant plants. 133e is an R2 plant derived from R33, which showed only partial resistance. (C) PVY titer in R2 transgenic plants following exposure to 35°C. Samples were taken 20 days after inoculation. V-3c and V-3j are resistant pGNIal.4-transformed plants (derivatives of V3 in A). 1-lOc is a pG3PVY3.9-transformed resistant plant (derivative of I10 in B). I-lj is an R2 plant, a progeny of a nonresistant pG3PVY3.9-transformed parent. VII-7 is a nonresistant plant transformed by the insertless vector pGA643. The active center of a potyvirus protease has been shown to reside in its C-terminal half, retaining activity after its N-terminal half is deleted (2, 24). NIa activity could not be followed in pGNIal.4-transformed plants lacking a substrate for processing; thus NIa activity was assumed from the cloned sequence, and a general protection mechanism by which excess inactive protease binds to the primary PVY polyprotein and prevents its correct processing is unlikely.

However, the possibility that some slight DNA modification accidentally took place, causing a few individual plants to express inactive PVY protease, can be neither ruled out nor easily verified. Nevertheless, these few plants may thereby become resistant. As only a small proportion of the ransformed plants exhibited protection, the inactive protease postulation may be applicable to these individual ncidents.

Several other observations regarding nonstructural viral genes are in line with this postulation. Plants transformed with the gene for the 54-kDa replicase read-through protein of TMV became resistant (23) as did those with the readthrough replicase of pea early browning virus (28). The induction ofTMV resistance by the 54-kDa putative replicase was later demonstrated to be translation-dependent (29). Anderson et al. (30) showed resistance in plants transformed with an inactive, truncated cucumber mosaic virus replicase sequence. This phenomenon supports the above-mentioned hypothesis that resistance conferred by nonstructural viral genes might result from overexpression of the defective viral protein, defined as "dominant negative mutation" by Herskowitz (31). We have recently obtained preliminary (as yet unpublished) data showing that a small proportion of the plants expressing the first three PVY cistrons, including the "helper component," are also resistant to PVY infection. If verified, these results broaden the spectrum of nonstructuralviral genes that engender resistance. This, in turn, argues for a more general mechanism (the dominant negative mutation?) as opposed to a specific mechanism for each individual gene. In certain cases, protection at the RNA level has been shown or implied. Marsh et al. (32) demonstrated that the introduction into protoplasts of mutated RNA from the replicase gene of brome mosaic virus interfered with the replication of the native RNA. Lindbo and Dougherty (33) demonstrated resistance in transgenic plants carrying untranslatable tobacco etch virus-CP sequences. A demonstration of expression of the PVY protease (as a protein) in the pGNIal.4 transformed plants was precluded for technical reasons. Hence, an RNA-mediated mechanism could also be postulated to explain the observed resistance. It is worth noting that NIa-transgenic plants maintained a signifcant degree of resistance after a considerable increase in temperature for relatively long periods.



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