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Cotton stands as a pillar in the textile industry due to its superior natural fibers. Lignin, a complex polymer synthesized from phenylalanine and deposited in mature cotton fibers, is believed to be essential for fiber quality, although the precise effects remain largely unclear. In this study, we characterized two ubiquitously expressed cinnamyl alcohol dehydrogenases (CAD), GhCAD37A and GhCAD37D (GhCAD37A/D), in Gossypium hirsutum. GhCAD37A/D possess CAD enzymatic activities, to catalyze the generation of monolignol products during lignin biosynthesis. Analysis of transgenic cotton knockout and overexpressing plants revealed that GhCAD37A/D are important regulators of fiber quality, positively impacting breaking strength but negatively affecting fiber length and elongation percentage by modulating lignin biosynthesis in fiber cells. Moreover, GhCAD37A/D are shown to modulate anther vitality and affect stem lodging trait in cotton by influencing lignin biosynthesis in the vascular bundles of anther and stem, respectively. Additionally, our study revealed that Ghcad37A/D knockout plants displayed red stem xylem, likely due to the overaccumulation of aldehyde intermediates in the phenylpropanoid metabolism pathway, as indicated by metabolomics analysis. Thus, our work illustrates that GhCAD37A/D are two important enzymes of lignin biosynthesis in different cotton organs, influencing fiber quality, anther vitality, and stem lodging., (© 2024 Society for Experimental Biology and John Wiley & Sons Ltd.)

Heat stress (HS) poses a significant challenge to plant survival, necessitating sophisticated molecular mechanisms to maintain cellular homeostasis. Here, we identify SICKLE (SIC) as a key modulator of HS responses in Arabidopsis (Arabidopsis thaliana). SIC is required for the sequestration of RNA DEBRANCHING ENZYME 1 (DBR1), a rate-limiting enzyme of lariat intronic RNA (lariRNA) decay, into stress granules (SGs). The sequestration of DBR1 by SIC enhances the accumulation of lariRNAs, branched circular RNAs derived from excised introns during pre-mRNA splicing, which in turn promote the transcription of their parental genes. Our findings further demonstrate that SIC-mediated DBR1 sequestration in SGs is crucial for plant HS tolerance, as deletion of the N-terminus of SIC (SIC 1-244 ) impairs DBR1 sequestration and compromises plant response to HS. Overall, our study unveils a mechanism of transcriptional regulation in the HS response, where lariRNAs are enriched through DBR1 sequestration, ultimately promoting the transcription of heat stress tolerance genes., (© 2024. The Author(s).)

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., Competing Interests: Conflict of interest statement. None declared., (© The Author(s) 2023. Published by Oxford University Press on behalf of American Society of Plant Biologists.)

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, Gβ, and Gγ. In Arabidopsis , the Gβγ dimer serves as a positive regulator of plant defense. Conversely, in species such as rice, maize, cotton, and tomato, mutants deficient in Gβ exhibit constitutively active defense responses, suggesting a contrasting negative role for Gβ in defense mechanisms within these plants. Using a transient overexpression approach in addition to knockout mutants, we observed that Gβγ 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 Gβγ 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.

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., Competing Interests: The authors declare no conflict of interest.

The availability of efficient diagnostic methods is crucial to monitor the incidence of crop diseases and implement effective management strategies. One of the most important elements in diagnostics, especially in large acreage crops, is the sampling strategy as hundreds of thousands of individual plants can grow in a single farm, making it difficult to assess disease incidence in field surveys. This problem is compounded when there are no external disease symptoms, as in the case for the ratoon stunting disease (RSD) in sugarcane. We have developed an alternative approach of disease surveillance by using the crude cane juice expressed at the sugar factory (mill). For this purpose, we optimized DNA extraction and amplification conditions for the bacterium Leifsonia xyli subsp xyli , the causal agent of RSD. The use of nucleic acid dipsticks and LAMP isothermal amplification allows to perform the assays at the mills, even in the absence of molecular biology laboratories. Our method has been validated using the qPCR industry standard and shows higher sensitivity. This approach circumvents sampling limitations, providing RSD incidence evaluation on commercial crops and facilitating disease mapping across growing regions. There is also potential is to extend the technology to other sugarcane diseases as well as other processed crops., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The author(s) declared that they were an editorial board member of Frontiers, at the time of submission. This had no impact on the peer review process and the final decision., (Copyright © 2023 Burman, Mason, Hintzsche, Zou, Gibbs, MacGillycuddy, Magarey and Botella.)

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., (© American Society of Plant Biologists 2022. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

Early detection of pests and pathogens is of paramount importance in reducing agricultural losses. One approach to early detection is point-of-care (POC) diagnostics, which can provide early warning and therefore allow fast deployment of preventive measures to slow down the establishment of crop diseases. Among the available diagnostic technologies, nucleic acid amplification-based diagnostics provide the highest sensitivity and specificity, and those technologies that forego the requirement for thermocycling show the most potential for use at POC. In this review, I discuss the progress, advantages, and disadvantages of the established and most promising POC amplification technologies. The success and usefulness of POC amplification are ultimately dependent on the availability of POC-friendly nucleic acid extraction methods and amplification readouts, which are also briefly discussed in the review.

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., (© 2022. The Author(s).)

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., (© 2022 The Authors. Plant, Cell & Environment published by John Wiley & Sons Ltd.)

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., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Wei, Liu, Wang, Luo, Chen, Li, Wang, Botella and Zhang.)

Leaf senescence is an evolutionarily acquired process and it is critical for plant fitness. During senescence, macromolecules and nutrients are disassembled and relocated to actively growing organs. Plant leaf senescence process can be triggered by developmental cues and environmental factors, proper regulation of this process is essential to improve crop yield. Protein kinases are enzymes that modify their substrates activities by changing the conformation, stability, and localization of those proteins, to play a crucial role in the leaf senescence process. Impressive progress has been made in understanding the role of different protein kinases in leaf senescence recently. This review focuses on the recent progresses in plant leaf senescence-related kinases. We summarize the current understanding of the function of kinases on senescence signal perception and transduction, to help us better understand how the orderly senescence degeneration process is regulated by kinases, and how the kinase functions in the intricate integration of environmental signals and leaf age information., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Yang, Miao, Liu, Botella, Li, Li and Song.)

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, β, 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 Gβγ 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.

Key Message: Endogenous U6 promoters increase CRISPR/Cas9 editing efficiency in sorghum and may be useful for gene editing applications in other cereals., (© 2021. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

CRISPR/Cas-based genome editing technologies have the potential to fast-track large-scale crop breeding programs. However, the rigid cell wall limits the delivery of CRISPR/Cas components into plant cells, decreasing genome editing efficiency. Established methods, such as Agrobacterium tumefaciens -mediated or biolistic transformation have been used to integrate genetic cassettes containing CRISPR components into the plant genome. Although efficient, these methods pose several problems, including 1) The transformation process requires laborious and time-consuming tissue culture and regeneration steps; 2) many crop species and elite varieties are recalcitrant to transformation; 3) The segregation of transgenes in vegetatively propagated or highly heterozygous crops, such as pineapple, is either difficult or impossible; and 4) The production of a genetically modified first generation can lead to public controversy and onerous government regulations. The development of transgene-free genome editing technologies can address many problems associated with transgenic-based approaches. Transgene-free genome editing have been achieved through the delivery of preassembled CRISPR/Cas ribonucleoproteins, although its application is limited. The use of viral vectors for delivery of CRISPR/Cas components has recently emerged as a powerful alternative but it requires further exploration. In this review, we discuss the different strategies, principles, applications, and future directions of transgene-free genome editing methods., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Gong, Cheng and Botella.)

Heterotrimeric G protein Gβ-deficient mutants in rice and maize display constitutive immune responses, whereas Arabidopsis Gβ 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 Gβ 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 Gβ 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 Gβ 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 Gβ knockouts do not necessarily reflect divergences in G protein-mediated defense mechanisms., Competing Interests: The Authors did not report any conflict of interest., (© 2021 The Authors. Plant Direct published by American Society of Plant Biologists and the Society for Experimental Biology and John Wiley & Sons Ltd.)

Heterotrimeric GTP-binding proteins (G proteins), consisting of Gα, Gβ and Gγ subunits, transduce signals from a diverse range of extracellular stimuli, resulting in the regulation of numerous cellular and physiological functions in Eukaryotes. According to the classic G protein paradigm established in animal models, the bound guanine nucleotide on a Gα subunit, either guanosine diphosphate (GDP) or guanosine triphosphate (GTP) determines the inactive or active mode, respectively. In plants, there are two types of Gα subunits: canonical Gα subunits structurally similar to their animal counterparts and unconventional extra-large Gα subunits (XLGs) containing a C-terminal domain homologous to the canonical Gα along with an extended N-terminal domain. Both Gα and XLG subunits interact with Gβγ dimers and regulator of G protein signalling (RGS) protein. Plant G proteins are implicated directly or indirectly in developmental processes, stress responses, and innate immunity. It is established that despite the substantial overall similarity between plant and animal Gα subunits, they convey signalling differently including the mechanism by which they are activated. This review emphasizes the unique characteristics of plant Gα subunits and speculates on their unique signalling mechanisms.

Variation in grain size, a major determinant of grain yield and quality in cereal crops, is determined by both the plant's genetic potential and the available assimilate to fill the grain in the absence of stress. This study investigated grain size variation in response to variation in assimilate supply in sorghum using a diversity panel (n = 837) and a backcross-nested association mapping population (n = 1421) across four experiments. To explore the effects of genetic potential and assimilate availability on grain size, the top half of selected panicles was removed at anthesis. Results showed substantial variation in five grain size parameters with high heritability. Artificial reduction in grain number resulted in a general increase in grain weight, with the extent of the increase varying across genotypes. Genome-wide association studies identified 44 grain size quantitative trait locus (QTL) that were likely to act on assimilate availability and 50 QTL that were likely to act on genetic potential. This finding was further supported by functional enrichment analysis and co-location analysis with known grain number QTL and candidate genes. RNA interference and overexpression experiments were conducted to validate the function of one of the identified gene, SbDEP1, showing that SbDEP1 positively regulates grain number and negatively regulates grain size by controlling primary branching in sorghum. Haplotype analysis of SbDEP1 suggested a possible role in racial differentiation. The enhanced understanding of grain size variation in relation to assimilate availability presented in this study will benefit sorghum improvement and have implications for other cereal crops., (© 2021 Society for Experimental Biology and John Wiley & Sons Ltd.)

The extra-large guanosine-5'-triphosphate (GTP)-binding protein 2, XLG2, is an unconventional Gα subunit of the Arabidopsis (Arabidopsis thaliana) heterotrimeric GTP-binding protein complex with a major role in plant defense. In vitro biochemical analyses and molecular dynamic simulations show that affinity of XLG2 for GTP is two orders of magnitude lower than that of the conventional Gα, AtGPA1. Here we tested the physiological relevance of GTP binding by XLG2. We generated an XLG2(T476N) variant with abolished GTP binding, as confirmed by in vitro GTPγS binding assay. Yeast three-hybrid, bimolecular fluorescence complementation, and split firefly-luciferase complementation assays revealed that the nucleotide-depleted XLG2(T476N) retained wild-type XLG2-like interactions with the Gβγ dimer and defense-related receptor-like kinases. Both wild-type and nucleotide-depleted XLG2(T476N) restored the defense responses against Fusarium oxysporum and Pseudomonas syringae compromised in the xlg2 xlg3 double mutant. Additionally, XLG2(T476N) was fully functional restoring stomatal density, root growth, and sensitivity to NaCl, but failed to complement impaired germination and vernalization-induced flowering. We conclude that XLG2 is able to function in a GTP-independent manner and discuss its possible mechanisms of action., (© American Society of Plant Biologists 2021. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

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., (Copyright © 2021 Elsevier Inc. All rights reserved.)

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., (© 2020 Institute of Botany, Chinese Academy of Sciences.)

The complexity of current nucleic acid isolation methods limits their use outside of the modern laboratory environment. Here, we describe a fast and affordable method to purify nucleic acids from animal, plant, viral and microbial samples using a cellulose-based dipstick. Nucleic acids can be purified by dipping in-house-made dipsticks into just three solutions: the extract (to bind the nucleic acids), a wash buffer (to remove impurities) and the amplification reaction (to elute the nucleic acids). The speed and simplicity of this method make it ideally suited for molecular applications, both within and outside the laboratory, including limited-resource settings such as remote field sites and teaching institutions. Detailed instructions for how to easily manufacture large numbers of dipsticks in house are provided. Using the instructions, readers can create more than 200 dipsticks in <30 min and perform dipstick-based nucleic acid purifications in 30 s.

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 Gl 2 e . 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., (© 2019 The Authors. Plant Biotechnology Journal published by Society for Experimental Biology and The Association of Applied Biologists and John Wiley & Sons Ltd.)

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., Competing Interests: The authors have declared that no competing interests exist.

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.

Aims: Current culture-based methods for detection and determination of Campylobacter levels on processed chickens takes at least 2 days. Here we sought to develop a new complete, low-cost and rapid (approximately 2·5 h) detection system requiring minimal operator input., Methods and Results: We observed a strong correlation between culture-based cell counts and our ability to detect either Campylobacter jejuni or Campylobacter coli by loop-mediated isothermal amplification from the same samples. This knowledge was used to develop a rapid and simple five-step assay to quantify Campylobacter, which was subsequently assessed for its specificity, reproducibility and accuracy in quantifying Campylobacter levels from processed chickens. The assay was found to be highly specific for C. jejuni and C. coli and was capable of distinguishing between samples that are either within or exceeding the industry set target of 6000 Campylobacter colony forming units (CFU) per carcass (equivalent to 12 CFU per ml of chicken rinse) with >90% accuracy relative to culture-based methods., Conclusions: Our method can reliably quantify Campylobacter counts of processed chickens with an accuracy comparable to culture-based assays but provides results within hours as opposed to days., Significance and Impact of the Study: The research presented here will help improve food safety by providing fast Campylobacter detection that will enable the implementation of real-time risk management strategies in poultry processing plants to rapidly test processed chickens and identify effective intervention strategies. This technology is a powerful tool that can be easily adapted for other organisms and thus could be highly beneficial for a broad range of industries., (© 2019 The Authors. Journal of Applied Microbiology published by John Wiley & Sons Ltd on behalf of Society for Applied Microbiology.)

Heterotrimeric guanine nucleotide-binding proteins (G proteins), which are composed of α, β, and γ subunits, are versatile, guanine nucleotide-dependent, molecular on-off switches. In animals and fungi, the exchange of GDP for GTP on Gα controls G protein activation and is crucial for normal cellular responses to diverse extracellular signals. The model plant Arabidopsis thaliana has a single canonical Gα subunit, AtGPA1. We found that, in planta, the constitutively active, GTP-bound AtGPA1(Q222L) mutant and the nucleotide-free AtGPA1(S52C) mutant interacted with Gβγ1 and Gβγ2 dimers with similar affinities, suggesting that G protein heterotrimer formation occurred independently of nucleotide exchange. In contrast, AtGPA1(Q222L) had a greater affinity than that of AtGPA1(S52C) for Gβγ3, suggesting that the GTP-bound conformation of AtGPA1(Q222L) is distinct and tightly associated with Gβγ3. Functional analysis of transgenic lines expressing either AtGPA1(S52C) or AtGPA1(Q222L) in the gpa1 -null mutant background revealed various mutant phenotypes that were complemented by either AtGPA1(S52C) or AtGPA1(Q222L). We conclude that, in addition to the canonical GDP-GTP exchange-dependent mechanism, plant G proteins can function independently of nucleotide exchange., (Copyright © 2019 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.)

Molecular based diagnostic methods rely on the amplification of pathogen DNA but naked eye visualization of results is still challenging. We present here a simple and highly reliable DNA amplification readout system for naked eye detection of isothermally or PCR amplified DNA in less than 30 seconds. This system utilizes spermine to precipitate DNA amplicons and initiate bridging flocculation of a mix of charcoal and diatomaceous earth particles in suspension. In the absence of amplification, the charcoal particles remain suspended resulting in a black, non-transparent colloid solution while positive samples in which DNA amplification has occurred can be identified within seconds as the particles flocculate and settle leaving a transparent liquid phase. We have coupled this method with our rapid dipstick DNA purification method and isothermal DNA amplification to create a simple four-step diagnostic system that can be preassembled to reduce unnecessary manipulation in the field. The method's simplicity, low cost, minimal equipment and clear presence/absence readout makes it ideal for rapid diagnostic testing in the laboratory and in situations where users have limited technical training or resources including high school science classes and field-based research., Competing Interests: The research generated in this manuscript has been used to file for a provisional patent application in Australia to protect the intellectual property that has arisen from this work (Applicant: The University of Queensland; Filing date: May 21, 2019). Prior to the submission of this manuscript, the technology has not been licensed or sold to any company and it is not currently being commercialized., (This journal is © The Royal Society of Chemistry.)

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., (Copyright © 2019 The Author. Published by Elsevier Inc. All rights reserved.)

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.

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., (© The Author(s) 2019. Published by Oxford University Press on behalf of China Science Publishing & Media Ltd.)

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 GA 12 to produce GA 12 16, 17-dihydro-16α-ol (DHGA 12 ). We show that DHGA 12 , a C 20 -GA has an atypical structure compared to known active GAs but can bind to the GA receptor (GID1c). DHGA 12 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.

The CRISPR/Cas9 system has been widely used for targeted genome editing in numerous plant species. In Arabidopsis , constitutive promoters usually result in a low efficiency of heritable mutation in the T1 generation. In this work, CRISPR/Cas9 gene editing efficiencies using different promoters to drive Cas9 expression were evaluated. Expression of Cas9 under the constitutive CaMV 35S promoter resulted in a 2.3% mutation rate in T1 plants and failed to produce homozygous mutations in the T1 and T2 generations. In contrast, expression of Cas9 under two cell division-specific promoters, YAO and CDC45 , produced mutation rates of 80.9% to 100% in the T1 generation with nonchimeric mutations in the T1 (4.4⁻10%) and T2 (32.5⁻46.1%) generations. The pCDC45 promoter was used to modify a previously reported multiplex CRISPR/Cas9 system, replacing the original constitutive ubiquitin promoter. The multi-pCDC45-Cas9 system produced higher mutation efficiencies than the multi-pUBQ-Cas9 system in the T1 generation (60.17% vs. 43.71%) as well as higher efficiency of heritable mutations (11.30% vs. 4.31%). Sextuple T2 homozygous mutants were identified from a construct targeting seven individual loci. Our results demonstrate the advantage of using cell division promoters for CRISPR/Cas9 gene editing applications in Arabidopsis , especially in multiplex applications.

Background: When developing CRISPR/Cas9 systems for crops, it is crucial to invest time characterizing the genome editing efficiency of the CRISPR/Cas9 cassettes, especially if the transformation system is difficult or time-consuming. Cotton is an important crop for the production of fiber, oil, and biofuel. However, the cotton stable transformation is usually performed using Agrobacterium tumefaciens taking between 8 and 12 months to generate T 0 plants. Furthermore, cotton is a heterotetraploid and targeted mutagenesis is considered to be difficult as many genes are duplicated in this complex genome. The application of CRISPR/Cas9 in cotton is severely hampered by the long and technically challenging genetic transformation process, making it imperative to maximize its efficiency., Results: In this study, we provide a new system to evaluate and validate the efficiency of CRISPR/Cas9 cassettes in cotton using a transient expression system. By using this system, we could select the most effective CRISPR/Cas9 cassettes before the stable transformation. We have also optimized the existing cotton CRISPR/Cas9 system to achieve vastly improved mutagenesis efficiency by incorporating an endogenous GhU6 promoter that increases sgRNA expression levels over the Arabidopsis AtU6 - 29 promoter. The 300 bp GhU6.3 promoter was cloned and validated using the transient expression system. When sgRNAs were expressed under the control of the GhU6.3 promoter in CRISPR/Cas9 cassettes, expression levels were 6-7 times higher than those provided by the AtU6 - 29 promoter and CRISPR/Cas9-mediated mutation efficiency was improved 4-6 times., Conclusions: This study provides essential improvements to maximize CRISPR/Cas9-mediated mutation efficiency by reducing risk and workload for the application of CRISPR/Cas9 approaches in the targeted mutagenesis of cotton.

Background: The CRISPR/Cas9 system, composed of a single-guide RNA for target recognition and a Cas9 protein for DNA cleavage, has the potential to revolutionize agriculture as well as medicine. Even though extensive work has been done to improve the gene editing activity of CRISPR/Cas9, little is known about the regulation of this bacterial system in eukaryotic host cells, especially at the post-transcriptional level., Results: Here, we evaluate the expression levels of the two CRISPR/Cas9 components and the gene editing efficiency in a set of Arabidopsis mutants involved in RNA silencing. We find that mutants defective in the post-transcriptional gene-silencing pathway display significantly higher Cas9 and sgRNA transcript levels, resulting in higher mutagenesis frequencies than wild-type controls. Accordingly, silencing of AGO1 by introduction of an AGO1-RNAi cassette into the CRISPR/Cas9 vector provides an increase in gene editing efficiency. Co-expression of the viral suppressor p19 from the tomato bushy stunt virus to suppress the plant RNA-silencing pathway shows a strong correlation between the severity of the phenotypic effects caused by p19 and the gene editing efficiency of the CRISPR/Cas9 system for two different target genes, AP1 and TT4., Conclusions: This system has useful practical applications in facilitating the detection of CRISPR/Cas9-induced mutations in T1 plants as well as the identification of transgene-free T2 plants by simple visual observation of the symptom severity caused by p19. Our study shows that CRISPR/Cas9 gene editing efficiency can be improved by reducing RNA silencing in plants.

Background: Genetically modified cotton accounts for 64% of the world's cotton growing area (22.3 million hectares). The genome sequencing of the diploid cotton progenitors Gossypium raimondii and Gossypium arboreum as well as the cultivated Gossypium hirsutum has provided a wealth of genetic information that could be exploited for crop improvement. Unfortunately, gene functional characterization in cotton is lagging behind other economically important crops due to the low efficiency, lengthiness and technical complexity of the available stable transformation methods. We present here a simple, fast and efficient method for the transient transformation of G . hirsutum that can be used for gene characterization studies., Results: We developed a transient transformation system for gene characterization in upland cotton. Using β-glucuronidase as a reporter for Agrobacterium -mediated transformation assays, we evaluated multiple transformation parameters such as Agrobacterium strain, bacterial density, length of co-cultivation, chemicals and surfactants, which can affect transformation efficiency. After the initial characterization, the Agrobacterium EHA105 strain was selected and a number of binary constructs used to perform gene characterization studies. 7-days-old cotton seedlings were co-cultivated with Agrobacterium and transient gene expression was observed 5 days after infection of the plants. Transcript levels of two different transgenes under the control of the cauliflower mosaic virus (CaMV) 35S promoter were quantified by real-time reverse transcription PCR (qRT-PCR) showing a 3-10 times increase over the levels observed in non-infected controls. The expression patterns driven by the promoters of two G . hirsutum genes as well as the subcellular localization of their corresponding proteins were studied using the new transient expression system and our observations were consistent with previously published results using Arabidopsis as a heterologous system., Conclusions: The  Agrobacterium -mediated transient transformation method is a fast and easy transient expression system enabling high transient expression and transformation efficiency in upland cotton seedlings. Our method can be used for gene functional studies such as promoter characterization and protein subcellular localization in cotton, obviating the need to perform such studies in a heterologous system such as Arabidopsis .

Background: The CRISPR/Cas9 system is being used for genome editing purposes by many research groups in multiple plant species. Traditional sequencing methods to identify homozygous mutants are time-consuming, laborious and expensive., Results: We have developed a method to screen CRISPR/Cas9-induced mutants through Mutation Sites Based Specific Primers Polymerase Chain Reaction (MSBSP-PCR). The MSBSP-PCR method was successfully used to identify homozygous/biallelic mutants in Nicotiana tabacum and Arabidopsis thaliana , and we speculate that it can be used for the identification of CRISPR/Cas9-induced mutants in other plant species. Compared to traditional sequencing methods, MSBSP-PCR is simpler, faster and cheaper., Conclusions: The MSBSP-PCR method is simple to implement and can save time and cost in the screening of CRISPR/Cas9-induced homozygous/biallelic mutants.

[This corrects the article DOI: 10.1371/journal.pbio.2003916.].

Background: Soil salinization is one of the major environmental constraints to plant growth and agricultural production worldwide. Signaling components involving calcium (Ca 2+ ) and the downstream calcium-dependent protein kinases (CPKs) play key roles in the perception and transduction of stress signals. However, the study of CPKs in cotton and their functions in response to salt stress remain unexplored., Results: A total of 98 predicted CPKs were identified from upland cotton (Gossypium hirsutum L. 'TM-1'), and phylogenetic analyses classified them into four groups. Gene family distribution studies have revealed the substantial impacts of the genome duplication events to the total number of GhCPKs. Transcriptome analyses showed a wide distribution of CPKs' expression among different organs. A total of 19 CPKs were selected for their rapid responses to salt stress at the transcriptional level, most of which were also incduced by the thylene-releasing chemical ethephon, suggesting a partal overlap of the salinity and ethylene responses. Silencing of 4 of the 19 CPKs (GhCPK8, GhCPK38, GhCPK54, and GhCPK55) severely compromised the basal cotton resistance to salt stress., Conclusions: Our genome-wide expression analysis of CPK genes from up-land cotton suggests that CPKs are involved in multiple developmental responses as well as the response to different abiotic stresses. A cluster of the cotton CPKs was shown to participate in the early signaling events in cotton responses to salt stress. Our results provide significant insights on functional analysis of CPKs in cotton, especially in the context of cotton adaptions to salt stress.

Diagnostic technologies for the detection of plant pathogens with point-of-care capability and high multiplexing ability are an essential tool in the fight to reduce the large agricultural production losses caused by plant diseases. The main desirable characteristics for such diagnostic assays are high specificity, sensitivity, reproducibility, quickness, cost efficiency and high-throughput multiplex detection capability. This article describes and discusses various DNA-based point-of care diagnostic methods for applications in plant disease detection. Polymerase chain reaction (PCR) is the most common DNA amplification technology used for detecting various plant and animal pathogens. However, subsequent to PCR based assays, several types of nucleic acid amplification technologies have been developed to achieve higher sensitivity, rapid detection as well as suitable for field applications such as loop-mediated isothermal amplification, helicase-dependent amplification, rolling circle amplification, recombinase polymerase amplification, and molecular inversion probe. The principle behind these technologies has been thoroughly discussed in several review papers; herein we emphasize the application of these technologies to detect plant pathogens by outlining the advantages and disadvantages of each technology in detail.

Nucleic acid amplification is a powerful molecular biology tool, although its use outside the modern laboratory environment is limited due to the relatively cumbersome methods required to extract nucleic acids from biological samples. To address this issue, we investigated a variety of materials for their suitability for nucleic acid capture and purification. We report here that untreated cellulose-based paper can rapidly capture nucleic acids within seconds and retain them during a single washing step, while contaminants present in complex biological samples are quickly removed. Building on this knowledge, we have successfully created an equipment-free nucleic acid extraction dipstick methodology that can obtain amplification-ready DNA and RNA from plants, animals, and microbes from difficult biological samples such as blood and leaves from adult trees in less than 30 seconds. The simplicity and speed of this method as well as the low cost and availability of suitable materials (e.g., common paper towelling), means that nucleic acid extraction is now more accessible and affordable for researchers and the broader community. Furthermore, when combined with recent advancements in isothermal amplification and naked eye DNA visualization techniques, the dipstick extraction technology makes performing molecular diagnostic assays achievable in limited resource settings including university and high school classrooms, field-based environments, and developing countries.

Rhizoctonia solani, the causal agent of rice sheath blight disease, causes significant losses worldwide as there are no cultivars providing absolute resistance to this fungal pathogen. We have used Host Delivered RNA Interference (HD-RNAi) technology to target two PATHOGENICITY MAP KINASE 1 (PMK1) homologues, RPMK1-1 and RPMK1-2, from R. solani using a hybrid RNAi construct. PMK1 homologues in other fungal pathogens are essential for the formation of appressorium, the fungal infection structures required for penetration of the plant cuticle, as well as invasive growth once inside the plant tissues and overall viability of the pathogen within the plant. Evaluation of transgenic rice lines revealed a significant decrease in fungal infection levels compared to non-transformed controls and the observed delay in disease symptoms was further confirmed through microscopic studies. Relative expression levels of the targeted genes, RPMK1-1 and RPMK1-2, were determined in R. solani infecting either transgenic or control lines with significantly lower levels observed in R. solani infecting transgenic lines carrying the HD-RNAi constructs. This is the first report demonstrating the effectiveness of HD-RNAi against sheath blight and offers new opportunities for durable control of the disease as it does not rely on resistance conferred by major resistance genes.

Genome editing is an important tool for gene functional studies as well as crop improvement. The recent development of the CRISPR/Cas9 system using single guide RNA molecules (sgRNAs) to direct precise double strand breaks in the genome has the potential to revolutionize agriculture. Unfortunately, not all sgRNAs are equally efficient and it is difficult to predict their efficiency by bioinformatics. In crops such as cotton ( Gossypium hirsutum L.), with labor-intensive and lengthy transformation procedures, it is essential to minimize the risk of using an ineffective sgRNA that could result in the production of transgenic plants without the desired CRISPR-induced mutations. In this study, we have developed a fast and efficient method to validate the functionality of sgRNAs in cotton using a transient expression system. We have used this method to validate target sites for three different genes GhPDS, GhCLA1 , and GhEF1 and analyzed the nature of the CRISPR/Cas9-induced mutations. In our experiments, the most frequent type of mutations observed in cotton cotyledons were deletions (∼64%). We prove that the CRISPR/Cas9 system can effectively produce mutations in homeologous cotton genes, an important requisite in this allotetraploid crop. We also show that multiple gene targeting can be achieved in cotton with the simultaneous expression of several sgRNAs and have generated mutations in GhPDS and GhEF1 at two target sites. Additionally, we have used the CRISPR/Cas9 system to produce targeted gene fragment deletions in the GhPDS locus. Finally, we obtained transgenic cotton plants containing CRISPR/Cas9-induced gene editing mutations in the GhCLA1 gene. The mutation efficiency was very high, with 80.6% of the transgenic lines containing mutations in the GhCLA1 target site resulting in an intense albino phenotype due to interference with chloroplast biogenesis.

Seed size and seed weight are major quality attributes and important determinants of yield that have been strongly selected for during crop domestication. Limited information is available about the genetic control and genes associated with seed size and weight in sorghum. This study identified sorghum orthologs of genes with proven effects on seed size and weight in other plant species and searched for evidence of selection during domestication by utilizing resequencing data from a diversity panel. In total, 114 seed size candidate genes were identified in sorghum, 63 of which exhibited signals of purifying selection during domestication. A significant number of these genes also had domestication signatures in maize and rice, consistent with the parallel domestication of seed size in cereals. Seed size candidate genes that exhibited differentially high expression levels in seed were also found more likely to be under selection during domestication, supporting the hypothesis that modification to seed size during domestication preferentially targeted genes for intrinsic seed size rather than genes associated with physiological factors involved in the carbohydrate supply and transport. Our results provide improved understanding of the complex genetic control of seed size and weight and the impact of domestication on these genes.