Rapid Avr9- and Cf-9-Dependent Activation of MAP Kinases in Tobacco Cell Cultures and Leaves: Convergence of Resistance Gene, Elicitor, Wound, and Salicylate Responses

Abstract

The Cf-9 resistance ( R ) gene from tomato confers resistance to the fungal pathogen Cladosporium fulvum expressing the corresponding, pathogen-derived avirulence gene product Avr9. To understand how an initial R/Avr recognition event is transmitted and triggers the induction of plant defenses, we investigated early Avr9/Cf-9-dependent activation of protein kinases in transgenic tobacco expressing the Cf-9 gene. We identified two protein kinases of 46 and 48 kD, using myelin basic protein as substrate, that became rapidly activated in a strictly gene-for-gene manner within 2 to 5 min after Avr9 elicitation in both Cf9 tobacco plants and derived cell cultures. Studies with pharmacological inhibitors and effectors revealed that Ca 2 ؉ influx and a phosphorylation event(s) are required for kinase activation, but neither enzyme is involved in the Avr9-dependent synthesis of active oxygen species. The activation of both kinases is achieved via post-translational mechanisms, and the activation but not inactivation step includes tyrosine phosphorylation. Using specific antibodies, we found that the 46-and 48-kD kinases were similiar to WIPK (for wound-induced protein kinase) and SIPK (for salicylic acid-induced protein kinase), two previously characterized mitogen-activated protein (MAP) kinases from tobacco. In addition, Cf9 tobacco plants and cell cultures showed an Avr9-dependent accumulation of the WIPK transcript. Cf9 tobacco suspension cultures are thus a unique system in which to analyze the earliest events in R gene function. These data indicate that (1) the R / Avr -mediated induction of plant defense is accomplished via several parallel signaling mechanisms, and (2) R/Avr-dependent signal transduction pathways are interlinked at MAP kinases with responses of plants not only to non-race-specific elicitors but also to abiotic stimuli, such as wounding and mechanical stress. INTRODUCTIONThe capacity of plants to stop the growth of pathogens and parasites depends on early warning of invasion, followed by the activation of defense mechanisms. Disease resistance ( R ) genes are part of the plant's surveillance system and, in so-called gene-for-gene interactions, confer resistance to pathogens that carry the corresponding avirulence ( Avr ) genes (Flor, 1971). The R gene product is generally considered as a receptor for the matching Avr protein (Staskawicz et al., 1995). Despite the isolation of an increasing number of plant R genes (see below), little is known about the signal transduction chains that follow the R/Avr recognition event. A different picture emerges from the analysis of plant responses after nonspecific elicitation with bacterial or fungal oligosaccharides, proteins, or peptides that are not part of a matching Avr / R gene pair. To date, only one receptor, a 70-kD transmembrane ␤ -glucan elicitor binding protein from soybean, has been characterized, and the corresponding gene has been cloned (Umemoto et al., 1997). In parsley, a 91-kD transmembrane protein, which binds the fungal protein elicitor fro...

A Harpin Binding Site in Tobacco Plasma Membranes Mediates Activation of the Pathogenesis-Related Gene HIN1 Independent of Extracellular Calcium but Dependent on Mitogen-Activated Protein Kinase Activity

“…In contrast, activation of SIPK by ␤-elicitins Zhang et al, 1998;Zhang and Klessig, 2000;our unpublished data) or the ␤-elicitin parasiticein (Zhang et al, 1998) was much more prolonged. Similarly prolonged activation of SIPK activity has been reported in tobacco cells treated with Trichoderma viride-derived xylanase (Suzuki et al, 1999) or in Cf-9-transformed tobacco cells treated with the race-specific C. fulvum elicitor AVR9 (Romeis et al, 1999). Moreover, elicitins but not harpin Psph induced prolonged activation of the 44-kD MAPK, WIPK, and another yet undefined tobacco MAPK of 40 kD (Zhang et al, 1998;Zhang and Klessig, 2000).…”

“…In those cases investigated, MAPK activation by elicitor was dependent on extracellular Ca 2ϩ (Ligterink et al, 1997;Lebrun-Garcia et al, 1998;Romeis et al, 1999;Suzuki et al, 1999;Fellbrich et al, 2000). Particularly, ␤-elicitin-induced SIPK activity Figure 6) and HIN1 expression ( Figure 4) were dependent on extracellular Ca 2ϩ .…”

“…This is novel in that it suggests the functional involvement of MAPK (SIPK) activity in plant defense activation. Romeis et al (1999) showed inhibition of elicitor-induced tobacco SIPK activity by another MAPKK inhibitor, PD98059, which, however, did not block the elicitation of defense-related responses, such as production of reactive oxygen species. Furthermore, PD98059 inhibited the activation of two MAPKs of 39 and 44 kD and of PR gene expression in harpin Psstreated Arabidopsis cells (Desikan et al, 1999), but because the Arabidopsis SIPK ortholog AtMPK6 is a 49-kD protein (Nühse et al, 2000), it is not certain if either one of the PD98059-sensitive MAPKs represented AtMPK6.…”

“…To causally link harpin Psph -induced MAPK activity and HIN1 expression in tobacco, we used the inhibitor of MAPK kinases (MAPKK) U0126 (Favata et al, 1998). This inhibitor is believed to be more specific and more active against MAPKK than inhibitor PD98059 (Favata et al, 1998), which was reported recently to block the MAPK pathway in Arabidopsis and tobacco (Desikan et al, 1999;Romeis et al, 1999). Although PD98059 blocks MAPKK activity by binding directly to the inactive (nonphosphorylated) form of the enzyme, U0126 does not affect MAPKK activation by phosphorylation but inhibits the activated (phosphorylated) MAPKK at the catalytic site (Favata et al, 1998).…”

“…Tobacco mosaic virus infection of tobacco plants (Zhang and Klessig, 1998a) or treatment of tobacco cells with elicitins Zhang et al, 1998) and Trichoderma viride -derived xylanase (Suzuki et al, 1999) stimulated transient activation of a 48-kD salicylic acid-inducible MAPK (SIPK) Klessig, 1997, 2000). When the Cladosporium fulvum -derived race-specific elicitor AVR9 was infiltrated into tobacco plants expressing the tomato resistance gene Cf-9 , both SIPK and another tobacco mosaic virus and elicitin-responsive tobacco MAPK, WIPK (for wounding-induced protein kinase) (Zhang and Klessig, 1998b), were activated (Romeis et al, 1999). Moreover, elicitor-responsive parsley SIPK and WIPK and Arabidopsis SIPK orthologous enzymes have been reported (Ligterink et al, 1997;Nühse et al, 2000;Scheel et al, 2000).…”