Proc. Nati. Acad. Sci. USA Vol. 87, pp. 1633-1637, March 1990 Biochemistry Host regulation of the cauliflower mosaic virus multiplication cycle (minichromosome/gene regulation/reverse transcription/Brassica susceptibility) SIMON N. COVEY*, DAVID S. TURNER, ANDREW P. Lucy, AND KEITH SAUNDERS Department of Virus Research, Agricultural and Food Research Council Institute of Plant Science Research, John Innes Institute, Colney Lane, Norwich NR4 7UH, United Kingdom Communicated by Myron K. Brakke, December 12, 1989 (receivedfor review August 20, 1989) ABSTRACT The DNA genome of cauliflower mosaic virus forms have been characterized; they can be purified from (CaMV) replicates in the cytoplasm of infected plant cells by infected tissue by phenol extraction in the absence of virion reverse transcription of an RNA template. Viral RNA is DNA because CaMV virions are very stable in the presence generated in the nucleus by transcription of an episomal ofphenol (13). A variety of CaMV DNA forms accumulate in minichromosome containing supercoiled DNA. We have as- infected turnip leaves, including single-stranded DNAs, a sessed the relative activities of the nuclear and cytoplasmic variety of hairpin molecules, and linear double-stranded phases of the CaMV multiplication cycle by monitoring unen- DNA forms with single-strand extensions ofthe minus strand capsidated viral DNA forms and polyadenylylated RNAs in (19, 21), structures consistent with their being generated by different organs of one host plant and in different host species. reverse transcription. The SC DNA of the CaMV minichro- Systemically infected leaves of a highly susceptible host, turnip mosome is present as a relatively minor component of the (Brassica rapa), contained abundant 35S RNA and 19S RNA unencapsidated DNA fraction from leaves actively engaged transcripts and unencapsidated reverse transcription products in virus multiplication. However, the SC DNA form accu- but relatively little supercoiled DNA. In contrast, supercoiled mulates in callus derived from infected leaf tissue grown in DNA accumulated in roots and other tissues of turnip plants vitro in which there is relatively little virus replication, of viral RNA. implying that not all host plant tissues can support the but without significant amounts steady-state complete CaMV multiplication cycle (22). We report here an Infected but asymptomatic leaves of a less susceptible CaMV investigation of the composition of viral RNA and DNA host, kohlrabi (Brassica oleracea), contained supercoiled DNA forms isolated from different plant organs of a highly sus- almost exclusively but negligible viral RNA and DNA products ceptible CaMV host, Brassica rapa (turnip) and from less of reverse transcription. An allotetraploid species, rape (Bras- susceptible Brassica species. We show that the patterns of sica napus), exhibited infection characteristics and minichro- CaMV transcription and reverse transcription are not the mosome expression levels intermediate between the other two same in all organs of one species or in different host species. species from which it was derived. We conclude that expression Our results suggest that host regulation of the CaMV multi- of the CaMV minichromosome is a key phase of the virus plication cycle is imposed upon the viral minichromosome, multiplication cycle, which is regulated differentially in organs possibly through specific transcription factors that exhibit of a highly susceptible host species. Furthermore, this regula- species variation, influencing host susceptibility to infection. tion exhibits genetic variation among different Brassica species and controls host susceptibility to CaMV infection. MATERIALS AND METHODS Cauliflower mosaic virus (CaMV) has a double-stranded DNA Plants and Virus. The following plant species representing genome generated by reverse transcription of an RNA inter- three major groups of Brassica (23) were used to propagate mediate (for reviews, see refs. 1-3). Virion DNA contains CaMV: Brassica rapa, L. (genome descriptor: aa) subspecies site-specific discontinuities resulting from the replicative pro- rapifera (turnip) cv. Just Right; Brassica oleracea, L. (ge- cess. At a relatively early stage in the CaMV multiplication nome descriptor: cc) subspecies gongylodes (kohlrabi) cv. cycle, the discontinuities are repaired, and a supercoiled (SC) Purple Vienna; Brassica napus (genome descriptor: aacc) molecular form is synthesized, probably in the nucleus where subspecies oleifera (spring rape) cv. Brutor. Plants were it is found. Association of the SC DNA with histone-like mechanically inoculated with CaMV isolate Cabb B-JI at the proteins to produce a minichromosome precedes transcription 3- to 4-leaf stage and grown under greenhouse conditions at directed by host RNA polymerase II (4). Two major RNAs are 18-220C in a 16-hr photoperiod. Plant parts were harvested 25 transcribed from the minichromosome (5-9). 35S RNA is the days postinoculation. Leaves of 3-10 cm were harvested template for reverse transcription and a possible polycistronic from plants of each species and taken at equivalent stages of mRNA. 19S RNA is the mRNA for the CaMV gene VI protein. development. Leaves from infected turnip plants 25 days Synthesis of these RNAs is controlled by viral transcription postinoculation were processed for callus culture, exactly as promoters that have characteristics typical ofother eukaryotic described by Rollo and Covey (22). Stem samples were taken promoters recognized by RNA polymerase II. Isolated CaMV from plants grown in rosette form and included the stem- promoter fragments have been used to drive the expression of proximal parts of the leaf petioles. Root samples were taken many different foreign genes in a variety ofnonhost transgenic from the main tap root. plants in a manner often referred to as constitutive (for review, Nucleic Acid Analysis. Total CaMV DNA was determined see ref. 10). by dot-blot analysis, as described by Rollo and Covey (22). Transcription ofthe CaMV genome in the nucleus precedes Cellular nucleic acid was isolated by phenol/chloroform replication in the cytoplasm. Reverse transcription generates extraction from various plant tissues as described by Hull and a complex population of DNA forms that accumulate as Covey (13). This procedure effects isolation of total host unencapsidated molecules (11-21). Many of these molecular nucleic and unencapsidated viral DNA and RNA. CaMV The publication costs of this article were defrayed in part by page charge Abbreviations: CaMV, cauliflower mosaic virus; SC, supercoiled payment. This article must therefore be hereby marked "advertisement" DNA; hp, hairpin. in accordance with 18 U.S.C. §1734 solely to indicate this fact. *To whom reprint requests should be addressed. 1633 Downloaded by guest on September 24, 2021 1634 Biochemistry: Covey et al. Proc. Natl. Acad. Sci. USA 87 (1990) virions are stable in phenol, and so virion DNA does not and 19S RNA, together with a characteristic background of occur in this fraction. Polyadenylylated RNA was separated heterogeneous-sized molecules (Fig. 2, lane A). The RNA from the DNA and other cellular RNAs as described by Plant isolated from stems was qualitatively similar to that from et al. (24). Viral unencapsidated DNA forms were analyzed leaves, although there was less of it (Fig. 2, lane B). How- by blot hybridization after two-dimensional agarose gel elec- ever, negligible CaMV-specific polyadenylylated RNA was trophoresis (19, 21). Cellular nucleic acid samples of 10 or 20 found in roots (Fig. 2, lane C) or in callus (Fig. 2, lane D), ,ug were loaded into a corner circular well of 1.1% agarose despite the abundance of CaMV SC DNA compared with slab gels. Electrophoresis at 1.25 V/cm for 20 hr was in 25 leaves (see Fig. 1). On long exposure ofautoradiograms, very mM Trisphosphoric acid buffer, pH 7.6, in the first dimen- low levels of largely heterogeneous CaMV RNA were found sion and in the second dimension, at 90° orientation to the in RNA preparations from infected roots and callus (data not first, in alkaline medium (30 mM NaOH/2 mM EDTA). Gels shown). were depurinated, alkali treated, and neutralized before CaMV Replication Cycle Intermediates Also Differ in Bras- Southern blotting. Gels for comparison contained equivalent sica Species Less Susceptible to Infection. From a survey of a amounts of cellular total DNA, and exposure of autoradio- range of Brassica species we have determined that host grams was regulated by comparing the intensity of standard response to CaMV infection falls into three broad categories quantities of size-marker DNA, unless otherwise stated in the (unpublished work). B. rapa (aa) variants exhibit very severe text. Gel electrophoresis of equal amounts (2 gg) of cellular symptoms of leaf chlorosis and plant stunting (28) and yield total polyadenylylated RNA and Northern blotting of CaMV- a relatively high titer of total CaMV DNA ('400 ng of total specific polyadenylylated RNA was done as described by CaMV DNA per g of tissue at 20 days postinoculation). In Covey et al. (11). Southern and Northern (RNA) blots were contrast, B. oleracea (cc) accumulates relatively little virus probed for CaMV-specific sequences by hybridization with (-2 ng of total CaMV DNA per g of tissue at 20 days 32P-labeled full-length cloned CaMV virion DNA. postinoculation) and exhibits very mild symptoms or no symptoms at all depending upon the subspecies. The allo- tetraploid species B. napus (aacc) shows an intermediate RESULTS response