Cryptogein (CRY), a protein secreted by Phytophthora cryptogea, causes necrosis on tobacco (Nicotiana tabacum) plants at the site of application (the stem or the roots) and also on distant leaves. Autoradiography of plantlets after root absorption of radioiodinated CRY demonstrated a rapid migration of the label to the leaf lamina via the veins. Using an anti-CRY antiserum, a CRY-related antigen was detected in the stem and leaves of CRY-treated plants at a distance from the site of application. This antigen had the same molecular weight as CRY and was detected in the leaves as early as 1 hour after stem treatment, i.e. long before necrosis was detectable. The antigen was also detected in plants inoculated with P. cryptogea. The distant location of the necrosis induced by the fungus or by CRY can be ascribed to the migration of this protein, which is toxic to tobacco cells. It is proposed that CRY, which also elicits defense reactions in tobacco, might contribute to the hypersensitive response of tobacco to P. cryptogea.The interaction between the phytopathogenic fungus Phytophthora cryptogea and the nonhost plant tobacco (Nicotiana tabacum) leads to an HR2 characterized by plant tissue necrosis and a restricted growth of the fungus (5). An unusual feature of this HR is that necrosis occurs not only at the inoculation court but also on the leaves at a distance from the fungus. In culture, P. cryptogea secretes a protein, CRY (mol wt 10,323), which causes the rapid death of tobacco cells and is an elicitor of phytoalexin accumulation (4,6,11,12). When applied to the wounded stem or roots of a tobacco plant, CRY induces necrosis within 24 h at the point of application and also on distant leaves (14). This raises two questions: (a) does this elicitor actually move within the plant; and (b) interaction, then it should be possible to detect this proteinaceous elicitor in noninfected tissues from inoculated plants.We have investigated the possibility that CRY migrates in tobacco by two approaches: (a) autoradiography of plantlets after absorption of radioiodinated CRY by the roots; and (b) immunodetection of the protein in the stem and the leaves after application of exogenous CRY to the stem or to the roots, or after stem-inoculation with P. cryptogea. In addition, the behavior of CRY was compared with that of CAP, a related protein produced by Phytophthora capsici, which has slightly different biological effects on tobacco (14). MATERIALS AND METHODSTobacco plants (Nicotiana tabacum cv Xanthi nc) grown in a greenhouse for 30 d were transferred into a growth chamber (240C, 16 h daylength), and CRY was applied 10 d later to the stem or to the roots. Stem treatments were as previously described (14): the plant was decapitated above the third highest fully expanded leaf, and the wounded stem received 2.5 ,ug of elicitor as a 20-,uL drop of aqueous solution or a plug taken from a malt-agar culture of Phytophthora cryptogea; only the two upper remaining leaves (Li and L2) were left attached. In root treatments, t...

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Ten monoclonal antibodies were selected from mice immunized with a highly purified elicitin secreted by Phytophthora cryptogea, termed cryptogein. These antibodies could be classified into five groups according to their cross-reactivity to heterologous elicitins from other Phytophthora species, from strict specificity (reacting solely with cryptogein) to broad reactivity (reacting with all four elicitins under study). When examined on BIA core (a real-time biospecific interaction analyser), these monoclonal antibodies were found to recognize at least three different epitopes on the cryptogein molecule. Their use in elicitin detection and quantification was optimized in several ELISA protocols. A mixed monoclonal-polydonal antibody, indirect DAS-ELISA procedure detected as little as 20 pg of purified elicitin per well (100/il). The four elicitins could be detected with the aid of one of couple of polyvalent reagents, whilst each one could be detected separately using appropriate monoclonal antibodies. These protocols have been used to detect elicitins secreted by Phytophthora spp. into culture medium as well as in planta following plant inoculation.

Ten monoclonal antibodies (McAbs) against cucumber mosaic virus (CMV) have been raised from fusion experiments performed after immunizing mice with different CMV antigens. Their reactivities with members of the three CMV serotypes, CMV-DTL, CMV-ToRS, and CMV-Co were tested in a double antibody sandwich format of enzyme immunosorbent assay (ELISA). Several of the McAbs were specific for different members of the CMV-DTL and CMV-ToRS groups while two allowed the detection of CMV-Co. By using a mixture of two McAbs, any member of the three major CMV serotypes could be detected in infected plant sap. One of the antibodies made it possible to discriminate between subunits and whole virions of CMV-D when it was used in ELISA simultaneously as coating antibody and as biotin-conjugate. McAbs were shown to be useful for quantifying the amount of CMV present in plant sap.

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