Plant innate immunity relies on the recognition of pathogen effector molecules by nucleotide-binding-leucine-rich repeat (NB-LRR) immune receptor families. Previously we have shown the N immune receptor, a member of TIR-NB-LRR family, indirectly recognizes the 50 kDa helicase (p50) domain of Tobacco mosaic virus (TMV) through its TIR domain. We have identified an N receptor-interacting protein, NRIP1, that directly interacts with both N's TIR domain and p50. NRIP1 is a functional rhodanese sulfurtransferase and is required for N to provide complete resistance to TMV. Interestingly, NRIP1 that normally localizes to the chloroplasts is recruited to the cytoplasm and nucleus by the p50 effector. As a consequence, NRIP1 interacts with N only in the presence of the p50 effector. Our findings show that a chloroplastic protein is intimately involved in pathogen recognition. We propose that N's activation requires a prerecognition complex containing the p50 effector and NRIP1.
Virus-induced gene silencing (VIGS) is a widely used, powerful technique for reverse genetics. VIGS vectors derived from the Tobacco rattle virus (TRV) are among the most popular for VIGS. We have developed a TRV RNA2 vector that allows the insertion of gene silencing fragments by ligation-independent cloning (LIC). This new vector has several advantages over previous vectors, particularly for applications involving the analysis of large numbers of sequences, since TRV-LIC vectors containing the desired insert are obtained with 100% efficiency. Importantly, this vector allows the high-throughput cloning of silencing fragments without the use of costly enzymes required for recombination, as is the case with GATEWAY-based vectors. We generated a collection of silencing vectors based on 400 tomato (Solanum lycopersicum) expressed sequence tags in this TRV-LIC background. We have used this vector to identify roles for SlMADS1 and its Nicotiana benthamiana homologs, NbMADS4-1 and -2 in flowering. We find that NbMADS4-1 and -2 act nonredundantly in floral development and silencing of either gene results in loss of organ identity. This TRV-LIC vector should be a valuable resource to the plant community.The last decade has seen an explosion in the availability of plant gene sequences. The genomes of the model species Arabidopsis (Arabidopsis thaliana) and rice (Oryza sativa) have both been sequenced, while those of tomato (Solanum lycopersicum) and maize (Zea mays) are currently being sequenced (Mueller et al., 2005; http://www.sgn.cornell.edu/about/tomato_project/; http://www.maizesequence.org/index.html). Large collections of ESTs have also been generated for a variety of species that are widely used for research purposes. Concomitant with the availability of this sequence information, many important aspects of plant growth and development have been analyzed by DNA microarrays, leading to the identification of numerous genes potentially involved in these processes. At this time then, the challenge to most plant biologists is to effectively mine this data to identify and characterize the genes and gene products that are critical to the crucial processes that have been investigated. This calls for techniques that start with a known DNA sequence and allow the determination of biological function. This approach is called reverse genetics and some of the most common methods for performing reverse genetic studies are based on RNA silencing.Although recently discovered, RNA silencing is a wellcharacterized, endogenous system for monitoring RNA inside a cell and eliminating foreign molecules or inhibiting mRNA translation (for review, see Brodersen and Voinnet, 2006). It is a homology-based process that uses small RNA fragments to identify targets for destruction or inhibition. RNA silencing is also indispensable for normal plant growth and development, regulating the expression of central genes in flowering, meristem identity, and other processes (Meins et al., 2005). In plants, RNA silencing plays critical roles in viral ...
Summary The plant innate immune response requires a rapid, global reprogramming of cellular processes. Here we employed two complementary proteomic methods, two-dimensional differential in-gel electrophoresis (2D-DIGE) and iTRAQ, to identify differentially regulated proteins early during a defense response. Besides defense-related proteins, the constituents of the largest category of up-regulated proteins were cytoplasmic- and endoplasmic reticulum (ER)-residing molecular chaperones. Silencing of ER-resident protein disulfide isomerases, NbERp57 and NbP5, and the calreticulins, NbCRT2 and NbCRT3, lead to a partial loss of N immune receptor-mediated defense against Tobacco mosaic virus (TMV). Furthermore, NbCRT2 and NbCRT3 are required for the expression of a novel induced receptor-like kinase (IRK). IRK is a plasma membrane-localized protein required for the N-mediated hypersensitive response programmed cell death (HR-PCD) and resistance to TMV. These data support a model in which ER-resident chaperones are required for the accumulation of membrane bound or secreted proteins that are necessary for innate immunity.
Programmed cell death (PCD) initiated at the pathogeninfected sites during the plant innate immune response is thought to prevent the development of disease. Here, we describe the identification and characterization of an ER-localized type IIB Ca 2 þ -ATPase (NbCA1) that function as a regulator of PCD. Silencing of NbCA1 accelerates viral immune receptor N-and fungal-immune receptor Cf9-mediated PCD, as well as non-host pathogen Pseudomonas syringae pv. tomato DC3000 and the general elicitor cryptogein-induced cell death. The accelerated PCD rescues loss-of-resistance phenotype of Rar1, HSP90-silenced plants, but not SGT1-silenced plants. Using a genetically encoded calcium sensor, we show that downregulation of NbCA1 results in the modulation of intracellular calcium signalling in response to cryptogein elicitor. We further show that NbCAM1 and NbrbohB function as downstream calcium decoders in N-immune receptormediated PCD. Our results indicate that ER-Ca 2 þ -ATPase is a component of the calcium efflux pathway that controls PCD during an innate immune response.
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