Conserved Role of Heterotrimeric G Proteins in Plant Defense and Cell Death Progression.

Programmed cell death (PCD) is a critical process in plant immunity, enabling the targeted elimination of infected cells to prevent the spread of pathogens. The tight regulation of PCD within plant cells is well-documented; however, specific mechanisms remain elusive or controversial. Heterotrimeric G proteins are multifunctional signaling elements consisting of three distinct subunits, Gα, Gß, and Gγ. In Arabidopsis, the Gßγ dimer serves as a positive regulator of plant defense. Conversely, in species such as rice, maize, cotton, and tomato, mutants deficient in Gß exhibit constitutively active defense responses, suggesting a contrasting negative role for Gß in defense mechanisms within these plants. Using a transient overexpression approach in addition to knockout mutants, we observed that Gßγ enhanced cell death progression and elevated the accumulation of reactive oxygen species in a similar manner across Arabidopsis, tomato, and Nicotiana benthamiana, suggesting a conserved G protein role in PCD regulation among diverse plant species. The enhancement of PCD progression was cooperatively regulated by Gßγ and one Gα, XLG2. We hypothesize that G proteins participate in two distinct mechanisms regulating the initiation and progression of PCD in plants. We speculate that G proteins may act as guardees, the absence of which triggers PCD. However, in Arabidopsis, this G protein guarding mechanism appears to have been lost in the course of evolution.

E3 ligases MAC3A and MAC3B ubiquitinate UBIQUITIN-SPECIFIC PROTEASE14 to regulate organ size in Arabidopsis.

The molecular mechanisms controlling organ size during plant development ultimately influence crop yield. However, a deep understanding of these mechanisms is still lacking. UBIQUITIN-SPECIFIC PROTEASE14 (UBP14), encoded by DA3, is an essential factor determining organ size in Arabidopsis (Arabidopsis thaliana). Here, we identified two suppressors of the da3-1 mutant phenotype, namely SUPPRESSOR OF da3-1 1 and 2 (SUD1 and SUD2), which encode the E3 ligases MOS4-ASSOCIATED COMPLEX 3A (MAC3A) and MAC3B, respectively. The mac3a-1 and mac3b-1 mutations partially suppressed the high ploidy level and organ size phenotypes observed in the da3-1 mutant. Biochemical analysis showed that MAC3A and MAC3B physically interacted with and ubiquitinated UBP14/DA3 to modulate its stability. We previously reported that UBP14/DA3 acts upstream of the B-type cyclin-dependent kinase CDKB1;1 and maintains its stability to inhibit endoreduplication and cell growth. In this work, MAC3A and MAC3B were found to promote the degradation of CDKB1;1 by ubiquitinating UBP14/DA3. Genetic analysis suggests that MAC3A and MAC3B act in a common pathway with UBP14/DA3 to control endoreduplication and organ size. Thus, our findings define a regulatory module, MAC3A/MAC3B-UBP14-CDKB1;1, that plays a critical role in determining organ size and endoreduplication in Arabidopsis.

A low-cost, portable, dual-function readout device for amplification-based point-of-need diagnostics.

IMPORTANCE: The first critical step in timely disease management is rapid disease identification, which is ideally on-site detection. Of all the technologies available for disease identification, nucleic acid amplification-based diagnostics are often used due to their specificity, sensitivity, adaptability, and speed. However, the modules to interpret amplification results rapidly, reliably, and easily in resource-limited settings at point-of-need (PON) are in high demand. Therefore, we developed a portable, low-cost, and easy-to-perform device that can be used for amplification readout at PON to enable rapid yet reliable disease identification by users with minimal training.

Insights into the molecular mechanisms of CRISPR/Cas9-mediated gene targeting at multiple loci in Arabidopsis.

Homologous recombination-mediated gene targeting (GT) enables precise sequence knockin or sequence replacement, and thus is a powerful tool for heritable precision genome engineering. We recently established a clustered regularly interspaced short palindromic repeats/clustered regularly interspaced short palindromic repeats-associated protein 9 (CRISPR/Cas9)-mediated approach for heritable GT in Arabidopsis (Arabidopsis thaliana), but its broad utility was not tested, and the underlying molecular mechanism was unclear. Here, we achieved precise GT at 14 out of 27 tested endogenous target loci using the sequential transformation approach and obtained vector-free GT plants by backcrossing. Thus, the sequential transformation GT method provides a broadly applicable technology for precise genome manipulation. We show that our approach generates heritable GT in the egg cell or early embryo of T1 Arabidopsis plants. Analysis of imprecise GT events suggested that single-stranded transfer DNA (T-DNA)/VirD2 complexes produced during the Agrobacterium (Agrobacterium tumefaciens) transformation process may serve as the donor templates for homologous recombination-mediated repair in the GT process. This study provides new insights into the molecular mechanisms of CRISPR/Cas9-mediated GT in Arabidopsis.

Expanding the Editing Window of Cytidine Base Editors With the Rad51 DNA-Binding Domain in Rice.

Recently developed base editors provide a powerful tool for plant research and crop improvement. Although a number of different deaminases and Cas proteins have been used to improve base editors the editing efficiency, and editing window are still not optimal. Fusion of a non-sequence-specific single-stranded DNA-binding domain (DBD) from the human Rad51 protein between Cas9 nickase and the deaminase has been reported to dramatically increase the editing efficiency and expand the editing window of base editors in the mammalian cell lines and mouse embryos. We report the use of this strategy in rice, by fusing a rice codon-optimized human Rad51 DBD to the cytidine base editors AncBE4max, AncBE4max-NG, and evoFERNY. Our results show that the addition of Rad51 DBD did not increase editing efficiency in the major editing window but the editing range was expanded in all the three systems. Replacing the human Rad51 DBD with the rice Rad51 DBD homolog also expanded the editing window effectively.

Overexpression of AHL9 accelerates leaf senescence in Arabidopsis thaliana.

BACKGROUND: Leaf senescence, the final stage of leaf growth and development, is regulated by numerous internal factors and environmental cues. Ethylene is one of the key senescence related hormones, but the underlying molecular mechanism of ethylene-induced leaf senescence remains poorly understood. RESULTS: In this study, we identified one AT-hook like (AHL) protein, AHL9, as a positive regulator of leaf senescence in Arabidopsis thaliana. Overexpression of AHL9 significantly accelerates age-related leaf senescence and promotes dark-induced leaf chlorosis. The early senescence phenotype observed in AHL9 overexpressing lines is inhibited by the ethylene biosynthesis inhibitor aminooxyacetic acid suggesting the involvement of ethylene in the AHL9-associated senescence. RNA-seq and quantitative reverse transcription PCR (qRT-PCR) data identified numerous senescence-associated genes differentially expressed in leaves of AHL9 overexpressing transgenic plants. CONCLUSIONS: Our investigation demonstrates that AHL9 functions in accelerating the leaf senescence process via ethylene synthesis or signalling.

Heterotrimeric G Protein Signaling in Abiotic Stress.

As sessile organisms, plants exhibit extraordinary plasticity and have evolved sophisticated mechanisms to adapt and mitigate the adverse effects of environmental fluctuations. Heterotrimeric G proteins (G proteins), composed of α, ß, and γ subunits, are universal signaling molecules mediating the response to a myriad of internal and external signals. Numerous studies have identified G proteins as essential components of the organismal response to stress, leading to adaptation and ultimately survival in plants and animal systems. In plants, G proteins control multiple signaling pathways regulating the response to drought, salt, cold, and heat stresses. G proteins signal through two functional modules, the Gα subunit and the Gßγ dimer, each of which can start either independent or interdependent signaling pathways. Improving the understanding of the role of G proteins in stress reactions can lead to the development of more resilient crops through traditional breeding or biotechnological methods, ensuring global food security. In this review, we summarize and discuss the current knowledge on the roles of the different G protein subunits in response to abiotic stress and suggest future directions for research.

Abscisic acid-induced cytoplasmic translocation of constitutive photomorphogenic 1 enhances reactive oxygen species accumulation through the HY5-ABI5 pathway to modulate seed germination.

Seed germination is a physiological process regulated by multiple factors. Abscisic acid (ABA) can inhibit seed germination to improve seedling survival under conditions of abiotic stress, and this process is often regulated by light signals. Constitutive photomorphogenic 1 (COP1) is an upstream core repressor of light signals and is involved in several ABA responses. Here, we demonstrate that COP1 is a negative regulator of the ABA-mediated inhibition of seed germination. Disruption of COP1 enhanced Arabidopsis seed sensitivity to ABA and increased reactive oxygen species (ROS) levels. In seeds, ABA induced the translocation of COP1 to the cytoplasm, resulting in enhanced ABA-induced ROS levels. Genetic evidence indicated that HY5 and ABI5 act downstream of COP1 in the ABA-mediated inhibition of seed germination. ABA-induced COP1 cytoplasmic localization increased HY5 and ABI5 protein levels in the nucleus, leading to increased expression of ABI5 target genes and ROS levels in seeds. Together, our results reveal that ABA-induced cytoplasmic translocation of COP1 activates the HY5-ABI5 pathway to promote the expression of ABA-responsive genes and the accumulation of ROS during ABA-mediated inhibition of seed germination. These findings enhance the role of COP1 in the ABA signal transduction pathway.

Tomato and cotton G protein beta subunit mutants display constitutive autoimmune responses.

Heterotrimeric G protein Gß-deficient mutants in rice and maize display constitutive immune responses, whereas Arabidopsis Gß mutants show impaired defense, suggesting the existence of functional differences between monocots and dicots. Using CRISPR/Cas9, we produced one hemizygous tomato line with a mutated SlGB1 Gß gene. Homozygous slgb1 knockout mutants exhibit all the hallmarks of autoimmune mutants, including development of necrotic lesions, constitutive expression of defense-related genes, and high endogenous levels of salicylic acid (SA) and reactive oxygen species, resulting in early seedling lethality. Virus-induced silencing of Gß in cotton reproduced the symptoms observed in tomato mutants, confirming that the autoimmune phenotype is not limited to monocot species but is also shared by dicots. Even though multiple genes involved in SA and ethylene signaling are highly induced by Gß silencing in tomato and cotton, co-silencing of SA or ethylene signaling components in cotton failed to suppress the lethal phenotype, whereas co-silencing of the oxidative burst oxidase RbohD can repress lethality. Despite the autoimmune response observed in slgb1 mutants, we show that SlGB1 is a positive regulator of the pathogen-associated molecular pattern (PAMP)-triggered immunity (PTI) response in tomato. We speculate that the phenotypic differences observed between Arabidopsis and tomato/cotton/rice/maize Gß knockouts do not necessarily reflect divergences in G protein-mediated defense mechanisms.

Rapid parasite detection utilizing a DNA dipstick.

Molecular diagnostics are powerful tools for disease detection but are typically confined to the laboratory environment due to the cumbersome methods required to extract nucleic acids from biological samples. Accurate diagnosis is essential for early detection of parasitic worm infections and for monitoring control programs, particularly during new transmission outbreaks to limit infection spread. We optimized the recently developed DNA dipstick technology to purify Schistosoma japonicum DNA from different life stages in <60 s. We successfully detected DNA from adult worms, eggs and infected snails. The speed and simplicity of this method enables the point-of-care detection of S. japonicum.

Genome editing for plant research and crop improvement.

The advent of clustered regularly interspaced short palindromic repeat (CRISPR) has had a profound impact on plant biology, and crop improvement. In this review, we summarize the state-of-the-art development of CRISPR technologies and their applications in plants, from the initial introduction of random small indel (insertion or deletion) mutations at target genomic loci to precision editing such as base editing, prime editing and gene targeting. We describe advances in the use of class 2, types II, V, and VI systems for gene disruption as well as for precise sequence alterations, gene transcription, and epigenome control.

ABC1K10a, an atypical kinase, functions in plant salt stress tolerance.

BACKGROUND: ABC1K (Activity of BC1 complex Kinase) is an evolutionarily primitive atypical kinase family widely distributed among prokaryotes and eukaryotes. The ABC1K protein kinases in Arabidopsis are predicted to localize either to the mitochondria or chloroplasts, in which plastid-located ABC1K proteins are involved in the response against photo-oxidative stress and cadmium-induced oxidative stress. RESULTS: Here, we report that the mitochondria-localized ABC1K10a functions in plant salt stress tolerance by regulating reactive oxygen species (ROS). Our results show that the ABC1K10a expression is induced by salt stress, and the mutations in this gene result in overaccumulation of ROS and hypersensitivity to salt stress. Exogenous application of the ROS-scavenger GSH significantly represses ROS accumulation and rescues the salt hypersensitive phenotype of abc1k10a. ROS overaccumulation in abc1k10a mutants under salt stress is likely due to the defect in mitochondria electron transport chain. Furthermore, defects of several other mitochondria-localized ABC1K genes also result in salt hypersensitivity. CONCLUSIONS: Taken together, our results reveal that the mitochondria-located ABC1K10a regulates mitochondrial ROS production and is a positive regulator of salt tolerance in Arabidopsis.

Evaluation and improvement of isothermal amplification methods for point-of-need plant disease diagnostics.

A number of isothermal DNA amplification technologies claim to be ideal for point-of-need (PON) applications as they enable reactions to be performed using a single-temperature heat source (e.g. water bath). Thus, we examined several isothermal amplification methods focusing on simplicity, cost, sensitivity and reproducibility to identify the most suitable method(s) for low resource PON applications. A number of methods were found unsuitable as they either involved multiple temperature incubations, were relatively expensive or required relatively large amounts target DNA for amplification. Among the methods examined, loop-mediated isothermal amplification (LAMP) and recombinase polymerase amplification (RPA) were found to be the most suitable for PON applications as they are both single step methods that provide highly sensitive and reproducible amplifications. The speed of LAMP reactions was greatly enhanced, up to 76%, with the addition of loop primers while the presence of swarm primers and the sequestration of free magnesium ions with nucleotides also enhanced the amplification speed. In contrast, we were unable to enhance RPA's performance from the original published literature. While both RPA and LAMP have some drawbacks, either isothermal technology can reliably be used for on-site diagnostics with minimal equipment.

The gland localized CGP1 controls gland pigmentation and gossypol accumulation in cotton.

Pigment glands, also known as black glands or gossypol glands, are specific for Gossypium spp. These glands strictly confine large amounts of secondary metabolites to the lysigenous cavity, leading to the glands' intense colour and providing defence against pests and pathogens. This study performed a comparative transcriptome analysis of glanded versus glandless cotton cultivars. Twenty-two transcription factors showed expression patterns associated with pigment glands and were characterized. Phenotypic screening of the genes, via virus-induced gene silencing, showed an apparent disappearance of pigmented glands after the silencing of a pair of homologous MYB-encoding genes in the A and D genomes (designated as CGP1). Further study showed that CGP1a encodes an active transcription factor, which is specifically expressed in the gland structure, while CGP1d encodes a non-functional protein due to a fragment deletion, which causes premature termination. RNAi-mediated silencing and CRISPR knockout of CGP1 in glanded cotton cultivars generated a glandless-like phenotype, similar to the dominant glandless mutant Gl2e . Microscopic analysis showed that CGP1 knockout did not affect gland structure or density, but affected gland pigmentation. The levels of gossypol and related terpenoids were significantly decreased in cgp1 mutants, and a number of gossypol biosynthetic genes were strongly down-regulated. CGP1 is located in the nucleus where it interacts with GoPGF, a critical transcription factor for gland development and gossypol synthesis. Our data suggest that CGP1 and GoPGF form heterodimers to control the synthesis of gossypol and other secondary metabolites in cotton.

Perspectives on the Application of Genome-Editing Technologies in Crop Breeding.

Most conventional and modern crop-improvement methods exploit natural or artificially induced genetic variations and require laborious characterization of the progenies of multiple generations derived from time-consuming genetic crosses. Genome-editing systems, in contrast, provide the means to rapidly modify genomes in a precise and predictable way, making it possible to introduce improvements directly into elite varieties. Here, we describe the range of applications available to agricultural researchers using existing genome-editing tools. In addition to providing examples of genome-editing applications in crop breeding, we discuss the technical and social challenges faced by breeders using genome-editing tools for crop improvement.

Beyond Light: Insights Into the Role of Constitutively Photomorphogenic1 in Plant Hormonal Signaling.

Light is an important environmental factor with profound effects in plant growth and development. Constitutively photomorphogenic1 (COP1) is a vital component of the light signaling pathway as a negative regulator of photomorphogenesis. Although the role of COP1 in light signaling has been firmly established for some time, recent studies have proven that COP1 is also a crucial part of multiple plant hormonal regulatory pathways. In this article, we review the available evidence involving COP1 in hormone signaling, its molecular mechanisms, and its contribution to the complicated regulatory network linking light and plant hormone signaling.

Biosynthesis of DHGA12 and its roles in Arabidopsis seedling establishment.

Seed germination and photoautotrophic establishment are controlled by the antagonistic activity of the phytohormones gibberellins (GAs) and abscisic acid (ABA). Here we show that Arabidopsis thaliana GAS2 (Gain of Function in ABA-modulated Seed Germination 2), a protein belonging to the Fe-dependent 2-oxoglutarate dioxygenase superfamily, catalyzes the stereospecific hydration of GA12 to produce GA12 16, 17-dihydro-16α-ol (DHGA12). We show that DHGA12, a C20-GA has an atypical structure compared to known active GAs but can bind to the GA receptor (GID1c). DHGA12 can promote seed germination, hypocotyl elongation and cotyledon greening. Silencing and over-expression of GAS2 alters the ABA/GA ratio and sensitivity to ABA during seed germination and photoautotrophic establishment. Hence, we propose that GAS2 acts to modulate hormonal balance during early seedling development.

Gene editing in plants: progress and challenges.

The clustered regularly interspaced short palindromic repeat (CRISPR)-associated protein 9 (Cas9) genome editing system is a powerful tool for targeted gene modifications in a wide range of species, including plants. Over the last few years, this system has revolutionized the way scientists perform genetic studies and crop breeding, due to its simplicity, flexibility, consistency and high efficiency. Considerable progress has been made in optimizing CRISPR/Cas9 systems in plants, particularly for targeted gene mutagenesis. However, there are still a number of important challenges ahead, including methods for the efficient delivery of CRISPR and other editing tools to most plants, and more effective strategies for sequence knock-ins and replacements. We provide our viewpoint on the goals, potential concerns and future challenges for the development and application of plant genome editing tools.