NopL, an effector protein of Rhizobium sp. NGR234, thwarts activation of plant defense reactions

Abstract

Bacterial effector proteins delivered into eukaryotic cells via bacterial type III secretion systems are important virulence factors in plant-pathogen interactions. Type III secretion systems have been found in Rhizobium species that form symbiotic, nitrogen-fixing associations with legumes. One such bacterium, Rhizobium sp. NGR234, secretes a number of type III effectors, including nodulation outer protein L (NopL, formerly y4xL). Here, we show that expression of nopL in tobacco (Nicotiana tabacum) prevents full induction of pathogenesis-related (PR) defense proteins. Transgenic tobacco plants that express nopL and were infected with potato virus Y (necrotic strain 605) exhibited only very low levels of chitinase (class I) and beta-1,3-glucanase (classes I and III) proteins. Northern-blot analysis indicated that expression of nopL in plant cells suppresses transcription of PR genes. Treatment with ethylene counteracted the effect of NopL on chitinase (class I). Transgenic Lotus japonicus plants that expressed nopL exhibited delayed development and low chitinase levels. In vitro experiments showed that NopL is a substrate for plant protein kinases. Together, these data suggest that NopL, when delivered into the plant cell, modulates the activity of signal transduction pathways that culminate in activation of PR proteins.

Figure 1.

Detection of NopL protein (M_r ≈ 37 kDa) in different transgenic tobacco lines that express nopL (NtNopL lines). Total soluble proteins (10 μg) from individual plants were analyzed by immunoblotting using antibodies raised against purified NopL protein. Purified NopL served as a control. Wt, Wild-type plants; C, control plants transformed with the vector pPZP112; NopL, NopL protein (1 μg) purified from Escherichia coli pPROEX-1nopL from which the 6xHis tag was removed (apparent M_r ≈ 37 kDa). L1 through L8, NtNopL lines 1 through 8 (tobacco lines expressing nopL).

Figure 2.

Tobacco plants that express nopL have a heightened susceptibility to potato virus Y N605. A, Virus accumulation in tobacco plants inoculated with potato virus Y N605 2 weeks after inoculation (six plants per line expressing nopL, 15 control plants transformed with the vector pPZP112, and 15 wild-type plants; all plants were grown individually in pots). An ELISA was used to quantify the degree of infection using antibodies directed against the virus coat protein that was conjugated to alkaline phosphatase. Values are means ± se of the signal expressed in relative units. Compared with wild-type plants, virus accumulation was significantly increased in plants that express nopL (L1, n = 21, χ²_[1] = 8.31, P = 0.004; L2, n = 21, χ²_[1] = 8.31, P = 0.004; L3, n = 21, χ²_[1] = 8.31, P = 0.004; L4, n = 19, χ²_[1] =5.4, P = 0.02; L5, n = 21, χ²_[1] = 8.31, P = 0.004). B, Fresh weight (aerial parts) of virus-infected plants 3 weeks after inoculation. Data show similar values, indicating normal growth of the NtNopL lines tested. Wt, Wild-type plants; C, control plants with the vector pPZP112; L1 through L5, NtNopL lines 1 through 5 (tobacco lines that express nopL).

Figure 3.

Defense reactions of tobacco plants that express nopL. Plants were infected with potato virus Y N605. Two weeks later, plant defense reactions were measured in virus-infected leaf discs harvested from the neighboring leaf. A, Chitinase (class I) protein. Bottom, Extracts (10 μg of protein) from leaves were subjected to SDS-PAGE, blotted onto polyvinylidene difluoride (PVDF) membranes, and immunodetected using a rabbit serum raised against tobacco chitinase (class I; M_r ≈ 32 kDa). Top, For each plant extract tested, the intensity of the band that correlated with the amount of immunolabeled protein was quantified using the Gene Genius Bioimaging system. Data (relative values) indicate means ± se (n = 6 for each NtNopL line, n = 15 for wild-type plants, and n = 15 for control plants with the vector pPZP112). B, β-1,3-Glucanase proteins. Bottom, Immunoblotting of protein extracts from NtNopL lines using an antiserum raised against tobacco β-1,3-glucanase class I (M_r ≈ 33 kD), which crossreacts with β-1,3-glucanase class III (M_r ≈ 35 kD), resulting in a double band. Top, Intensity of the β-1,3-glucanase spots quantified with the Gene Genius Bioimaging system. C, Peroxidase activity, as measured using guaiacol as the substrate. Values indicate means ± se of enzyme activity per total soluble protein content in the extract (n = 6). Wt, Wild-type plants; C, control plants containing the vector pPZP112; L1 through L5, NtNopL lines 1 through 5 (tobacco lines expressing nopL); (+), infected with potato virus Y N605; (–), noninoculated control.

Figure 4.

Expression of nopL in tobacco inhibits transcription of class I chitinase. RNA isolated from virus-infected leaf discs (2 weeks p.i.) was hybridized against DNA of clone CHN50 that encodes tobacco class I chitinase. Ethidium bromide staining of the agarose gel confirmed equal loading of RNA. Wt, Wild-type plants; C, control plants containing the vector pPZP112; L1 through L5, NtNopL lines 1 through 5 (tobacco lines expressing nopL); (+), infected with potato virus Y N605; (–), noninoculated control.

Figure 5.

Effect of ethylene on induction of class I chitinase in tobacco. Tobacco plants were incubated in a closed chamber containing 100 μL L^–1 ethylene for 48 h, whereas control plants were incubated for the same period in another chamber without ethylene. After treatment, induction of chitinase (class I) was analyzed on immunoblots. A, Wild-type plants. B, Plants that express nopL. L1, Line NtNopL(1); L3, line NtNopL(3); (+E), treated with ethylene; (–E), control without ethylene.

Figure 6.

Detection of NopL and chitinase in different transgenic L. japonicus lines that express nopL (LjNopL lines). A, Protein extracts (10 μg) from 4-week-old L. japonicus plants were used for immunodetection with an antiserum directed against the NopL protein (M_r ≈ 37 kD). B, A chitinase (M_r ≈ 32 kD) of L. japonicus nodulated with NGR234 crossreacted with the antiserum raised against tobacco chitinase (class I). Wt, Wild-type plants; C, control plants with the pPZP112 vector; L1 through L3, LjNopL lines 1 through 3 (L. japonicus lines expressing nopL).

Figure 7.

NopL is a substrate for plant protein kinases. A, In vitro phosphorylation assay. Recombinant NopL (1 μg) purified from E. coli pPROEX-1nopL (carrying a 6x-His tag; apparent size of ≈40 kD) was incubated with a tobacco extract (10 μg of soluble protein) in the presence of [γ-³³P]ATP (see “Materials and Methods”). The reaction mixture was separated by SDS-PAGE and the dried gel was exposed to an x-ray-sensitive film. C, Control reaction containing the corresponding fraction purified from E. coli pPROEX-1 (without nopL). B, Immunoprecipitation of phosphorylated NopL. Recombinant NopL protein was radioactively labeled with the protein kinase assay and was then incubated with the antiserum directed against NopL. Immunocomplexes were precipitated with protein-A agarose beads. The precipitated NopL protein was analyzed by SDS-PAGE followed by autoradiography. NopL, Reaction with NopL protein and antiserum against NopL; C1, control reaction without NopL protein (using the protein fraction from E. coli pPROEX-1); C2, reaction with control rabbit serum.