Despite the large number of leucine-rich-repeat (LRR) receptor-like-kinases (RLKs) in plants and their conceptual relevance in signaling events, functional information is restricted to a few family members. Here we describe the characterization of new LRR-RLK family members as virulence targets of the geminivirus nuclear shuttle protein (NSP). NSP interacts specifically with three LRR-RLKs, NIK1, NIK2, and NIK3, through an 80-amino acid region that encompasses the kinase active site and A-loop. We demonstrate that these NSP-interacting kinases (NIKs) are membrane-localized proteins with biochemical properties of signaling receptors. They behave as authentic kinase proteins that undergo autophosphorylation and can also phosphorylate exogenous substrates. Autophosphorylation occurs via an intermolecular event and oligomerization precedes the activation of the kinase. Binding of NSP to NIK inhibits its kinase activity in vitro, suggesting that NIK is involved in antiviral defense response. In support of this, infectivity assays showed a positive correlation between infection rate and loss of NIK1 and NIK3 function. Our data are consistent with a model in which NSP acts as a virulence factor to suppress NIK-mediated antiviral responses.

The binding protein (BiP) is an important component of endoplasmic reticulum stress response of cells. Despite extensive studies in cultured cells, a protective function of BiP against stress has not yet been demonstrated in whole multicellular organisms. Here, we have obtained transgenic tobacco (Nicotiana tabacum L. cv Havana) plants constitutively expressing elevated levels of BiP or its antisense cDNA to analyze the protective role of this endoplasmic reticulum lumenal stress protein at the whole plant level. Elevated levels of BiP in transgenic sense lines conferred tolerance to the glycosylation inhibitor tunicamycin during germination and tolerance to water deficit during plant growth. Under progressive drought, the leaf BiP levels correlated with the maintenance of the shoot turgidity and water content. The protective effect of BiP overexpression against water stress was disrupted by expression of an antisense BiP cDNA construct. Although overexpression of BiP prevented cellular dehydration, the stomatal conductance and transpiration rate in droughted sense leaves were higher than in control and antisense leaves. The rate of photosynthesis under water deficit might have caused a degree of greater osmotic adjustment in sense leaves because it remained unaffected during water deprivation, which was in marked contrast with the severe drought-induced decrease in the CO 2 assimilation in control and antisense leaves. In antisense plants, the water stress stimulation of the antioxidative defenses was higher than in control plants, whereas in droughted sense leaves an induction of superoxide dismutase activity was not observed. These results suggest that overexpression of BiP in plants may prevent endogenous oxidative stress.

Plants and plant pathogens are subject to continuous co-evolutionary pressure for dominance, and the outcomes of these interactions can substantially impact agriculture and food security1–3. In virus– plant interactions, one of the major mechanisms for plant antiviral immunity relies on RNA silencing, which is often suppressed by co-evolving virus suppressors, thus enhancing viral pathogenicity in susceptible hosts1. In addition, plants use the nucleotide-binding and leucine-rich repeat (NB-LRR) domain-containing resistance proteins, which recognize viral effectors to activate effector-triggered immunity in a defence mechanism similar to that employed in non-viral infections2,3. Unlike most eukaryotic organisms, plants are not known to activate mechanisms of host global translation suppression to fight viruses1,2. Here we demonstrate in Arabidopsis that the constitutive activation of NIK1, a leucine-rich repeat receptor-like kinase (LRR-RLK) identified as a virulence target of the begomovirus nuclear shuttle protein (NSP)4–6, leads to global translation suppression and translocation of the downstream component RPL10 to the nucleus, where it interacts with a newly identified MYB-like protein, L10-INTERACTING MYB DOMAIN-CONTAINING PROTEIN (LIMYB), to downregulate translational machinery genes fully. LIMYB overexpression represses ribosomal protein genes at the transcriptional level, resulting in protein synthesis inhibition, decreased viral messenger RNA association with polysome fractions and enhanced tolerance to begomovirus. By contrast, the loss of LIMYB function releases the repression of translation-related genes and increases susceptibility to virus infection. Therefore, LIMYB links immune receptor LRR-RLK activation to global translation suppression as an antiviral immunity strategy in plants.

The ER-resident molecular chaperone BiP (binding protein) was overexpressed in soybean. When plants growing in soil were exposed to drought (by reducing or completely withholding watering) the wild-type lines showed a large decrease in leaf water potential and leaf wilting, but the leaves in the transgenic lines did not wilt and exhibited only a small decrease in water potential. During exposure to drought the stomata of the transgenic lines did not close as much as in the wild type, and the rates of photosynthesis and transpiration became less inhibited than in the wild type. These parameters of drought resistance in the BiP overexpressing lines were not associated with a higher level of the osmolytes proline, sucrose, and glucose. It was also not associated with the typical drought-induced increase in root dry weight. Rather, at the end of the drought period, the BiP overexpressing lines had a lower level of the osmolytes and root weight than the wild type. The mRNA abundance of several typical drought-induced genes [NAC2, a seed maturation protein (SMP), a glutathione-S-transferase (GST), antiquitin, and protein disulphide isomerase 3 (PDI-3)] increased in the drought-stressed wild-type plants. Compared with the wild type, the increase in mRNA abundance of these genes was less (in some genes much less) in the BiP overexpressing lines that were exposed to drought. The effect of drought on leaf senescence was investigated in soybean and tobacco. It had previously been reported that tobacco BiP overexpression or repression reduced or accentuated the effects of drought. BiP overexpressing tobacco and soybean showed delayed leaf senescence during drought. BiP antisense tobacco plants, conversely, showed advanced leaf senescence. It is concluded that BiP overexpression confers resistance to drought, through an as yet unknown mechanism that is related to ER functioning. The delay in leaf senescence by BiP overexpression might relate to the absence of the response to drought.

NRPs (N-rich proteins) were identified as targets of a novel adaptive pathway that integrates endoplasmic reticulum (ER)and osmotic stress signals based on coordinate regulation and synergistic up-regulation by tunicamycin and polyethylene glycol treatments. This integrated pathway diverges from the molecular chaperone-inducing branch of the unfolded protein response (UPR) in several ways. While UPR-specific targets were inversely regulated by ER and osmotic stresses, NRPs required both signals for full activation. Furthermore, BiP (binding protein) overexpression in soybean prevented activation of the UPR by ER stress inducers, but did not affect activation of NRPs. We also found that this integrated pathway transduces a PCD signal generated by ER and osmotic stresses that result in the appearance of markers associated with leaf senescence. Overexpression of NRPs in soybean protoplasts induced caspase-3-like activity and promoted extensive DNA fragmentation. Furthermore, transient expression of NRPs in planta caused leaf yellowing, chlorophyll loss, malondialdehyde production, ethylene evolution, and induction of the senescence marker gene CP1. This phenotype was alleviated by the cytokinin zeatin, a potent senescence inhibitor. Collectively, these results indicate that ER stress induces leaf senescence through activation of plant-specific NRPs via a novel branch of the ER stress response.