The rice (Oryza sativa) spotted leaf11 (spl11) mutant was identified from an ethyl methanesulfonate-mutagenized indica cultivar IR68 population and was previously shown to display a spontaneous cell death phenotype and enhanced resistance to rice fungal and bacterial pathogens. Here, we have isolated Spl11 via a map-based cloning strategy. The isolation of the Spl11 gene was facilitated by the identification of three additional spl11 alleles from an IR64 mutant collection. The predicted SPL11 protein contains both a U-box domain and an armadillo (ARM) repeat domain, which were demonstrated in yeast and mammalian systems to be involved in ubiquitination and protein-protein interactions, respectively. Amino acid sequence comparison indicated that the similarity between SPL11 and other plant U-box-ARM proteins is mostly restricted to the U-box and ARM repeat regions. A single base substitution was detected in spl11, which results in a premature stop codon in the SPL11 protein. Expression analysis indicated that Spl11 is induced in both incompatible and compatible riceblast interactions. In vitro ubiquitination assay indicated that the SPL11 protein possesses E3 ubiquitin ligase activity that is dependent on an intact U-box domain, suggesting a role of the ubiquitination system in the control of plant cell death and defense.

Ubiquitination plays important roles in plant hormone signal transduction. We show that the RING finger E3 ligase, Arabidopsis thaliana SALT-AND DROUGHT-INDUCED RING FINGER1 (SDIR1), is involved in abscisic acid (ABA)-related stress signal transduction. SDIR1 is expressed in all tissues of Arabidopsis and is upregulated by drought and salt stress, but not by ABA. Plants expressing the ProSDIR1-b-glucuronidase (GUS) reporter construct confirmed strong induction of GUS expression in stomatal guard cells and leaf mesophyll cells under drought stress. The green fluorescent protein-SDIR1 fusion protein is colocalized with intracellular membranes. We demonstrate that SDIR1 is an E3 ubiquitin ligase and that the RING finger conservation region is required for its activity. Overexpression of SDIR1 leads to ABA hypersensitivity and ABA-associated phenotypes, such as salt hypersensitivity in germination, enhanced ABA-induced stomatal closing, and enhanced drought tolerance. The expression levels of a number of key ABA and stress marker genes are altered both in SDIR1 overexpression and sdir1-1 mutant plants. Cross-complementation experiments showed that the ABA-INSENSITIVE5 (ABI5), ABRE BINDING FACTOR3 (ABF3), and ABF4 genes can rescue the ABA-insensitive phenotype of the sdir1-1 mutant, whereas SDIR1 could not rescue the abi5-1 mutant. This suggests that SDIR1 acts upstream of those basic leucine zipper family genes. Our results indicate that SDIR1 is a positive regulator of ABA signaling.

NAC family genes encode plant-specific transcription factors involved in diverse biological processes. In this study, the Arabidopsis NAC gene ATAF1 was found to be induced by drought, high-salinity, abscisic acid (ABA), methyl jasmonate, mechanical wounding, and Botrytis cinerea infection. Significant induction of ATAF1 was found in an ABA-deficient mutant aba2 subjected to drought or high salinity, revealing an ABA-independent mechanism of expression. Arabidopsis ATAF1-overexpression lines displayed many altered phenotypes, including dwarfism and short primary roots. Furthermore, in vivo experiments indicate that ATAF1 is a bona fide regulator modulating plant responses to many abiotic stresses and necrotrophic-pathogen infection. Overexpression of ATAF1 in Arabidopsis increased plant sensitivity to ABA, salt, and oxidative stresses. Especially, ATAF1 overexpression plants, but not mutant lines, showed remarkably enhanced plant tolerance to drought. Additionally, ATAF1 overexpression enhanced plant susceptibility to the necrotrophic pathogen B. cinerea, but did not alter disease symptoms caused by avirulent or virulent strains of P. syringae pv tomato DC3000. Transgenic plants overexpressing ATAF1 were hypersensitive to oxidative stress, suggesting that reactive oxygen intermediates may be related to ATAF1-mediated signaling in response to both pathogen and abiotic stresses.

Previous analysis of transcriptional changes after elicitation of Cf-9 transgenic tobacco (Nicotiana tabacum) by Avr9 peptide revealed a rapidly upregulated gene, ACRE276. We show that ACRE276 is transiently induced in wounded leaves within 15 min, but upon Avr9 elicitor treatment, this upregulation is enhanced and maintained until cell death onset in Cf-9 tobacco. ACRE276 RNA interference (RNAi) silencing in tobacco results in loss of hypersensitive response (HR) specified by Cf resistance genes. ACRE276 RNAi plants are also compromised for HR mediated by the tobacco mosaic virus defense elicitor p50. Silencing tomato (Lycopersicon esculentum) ACRE276 leads to breakdown of Cf-9-specified resistance against Cladosporium fulvum leaf mold. We confirmed that tobacco ACRE276 is an E3 ubiquitin ligase requiring an intact U-box domain. Bioinformatic analyses revealed Arabidopsis thaliana PLANT U-BOX17 (PUB17) and Brassica napus ARC1 as the closest homologs of tobacco ACRE276. Transiently expressing PUB17 in Cf-9 tobacco silenced for ACRE276 restores HR, while mutant PUB17 lacking E3 ligase activity fails to do so, demonstrating that PUB17 ligase activity is crucial for defense signaling. Arabidopsis PUB17 knockout plants are compromised in RPM1-and RPS4-mediated resistance against Pseudomonas syringae pv tomato containing avirulence genes AvrB and AvrRPS4, respectively. We identify a conserved class of U-box ARMADILLO repeat E3 ligases that are positive regulators of cell death and defense across the Solanaceae and Brassicaceae.

Colorimetric gene detection based on gold nanoparticles (AuNPs) is an attractive detection format due to its simplicity. Here, we report a new design for a colorimetric gene-sensing platform based on the CRISPR/Cas system that has improved specificity, sensitivity, and universality. CRISPR/Cas12a and CRISPR/Cas13a have two distinct catalytic activities and are used for specific target gene recognition. Programmable recognition of DNA by Cas12a/crRNA and RNA by Cas13a/crRNA with a complementary sequence activates the nonspecific trans-ssDNA or -RNA cleavage, respectively, thus degrading the ssDNA or RNA linkers which are designed as a hybridization template for the AuNP-DNA probe pair. Target-induced trans -ssDNA or RNA cleavage leads to a distance-dependent color change for the AuNP-DNA probe pair. In this platform, naked eye detection of transgenic rice, African swine fever virus (ASFV), and a miRNA can be completed within 1 hour. Our colorimetric gene-sensing method shows superior characteristics, such as probe universality, isothermal reaction conditions, on-site detection capability, and sensitivity that is comparable to that of the fluorescent detection; thus, this method represents a robust next generation gene detection platform.