Changing the diagnostic paradigm for sugarcane: development of a mill-based diagnostic for ratoon stunting disease in crude cane juice.

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.

Point-of-Care DNA Amplification for Disease Diagnosis and Management.

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.

Function of Protein Kinases in Leaf Senescence of Plants.

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.

Endogenous U6 promoters improve CRISPR/Cas9 editing efficiencies in Sorghum bicolor and show potential for applications in other cereals.

KEY MESSAGE: Endogenous U6 promoters increase CRISPR/Cas9 editing efficiency in sorghum and may be useful for gene editing applications in other cereals.

Heterotrimeric G Proteins in Plants: Canonical and Atypical Gα Subunits.

Heterotrimeric GTP-binding proteins (G proteins), consisting of Gα, Gß 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 Gßγ 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.

Manipulating assimilate availability provides insight into the genes controlling grain size in sorghum.

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.

GTP binding by Arabidopsis extra-large G protein 2 is not essential for its functions.

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 Gßγ 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.

Non-GM Genome Editing Approaches in Crops.

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.

Rapid (30-second), equipment-free purification of nucleic acids using easy-to-make dipsticks.

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.

Nucleotide exchange-dependent and nucleotide exchange-independent functions of plant heterotrimeric GTP-binding proteins.

Heterotrimeric guanine nucleotide-binding proteins (G proteins), which are composed of α, ß, 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 Gßγ1 and Gßγ2 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 Gßγ3, suggesting that the GTP-bound conformation of AtGPA1(Q222L) is distinct and tightly associated with Gßγ3. 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.

A transient transformation system for gene characterization in upland cotton (Gossypium hirsutum).

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 ß-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.

A simple and cost-effective method for screening of CRISPR/Cas9-induced homozygous/biallelic mutants.

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.

Whole-Genome Analysis of Candidate genes Associated with Seed Size and Weight in Sorghum bicolor Reveals Signatures of Artificial Selection and Insights into Parallel Domestication in Cereal Crops.

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.

Genome Editing in Cotton with the CRISPR/Cas9 System.

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.

Host Delivered RNAi, an efficient approach to increase rice resistance to sheath blight pathogen (Rhizoctonia solani).

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.

Novel Chitinase Gene LOC_Os11g47510 from Indica Rice Tetep Provides Enhanced Resistance against Sheath Blight Pathogen Rhizoctonia solani in Rice.

Sheath blight disease (ShB), caused by the fungus Rhizoctonia solani Kühn, is one of the most destructive diseases of rice (Oryza sativa L.), causing substantial yield loss in rice. In the present study, a novel rice chitinase gene, LOC_Os11g47510 was cloned from QTL region of R. solani tolerant rice line Tetep and used for functional validation by genetic transformation of ShB susceptible japonica rice line Taipei 309 (TP309). The transformants were characterized using molecular and functional approaches. Molecular analysis by PCR using a set of primers specific to CaMv 35S promoter, chitinase and HptII genes confirmed the presence of transgene in transgenic plants which was further validated by Southern hybridization. Further, qRT-PCR analysis of transgenic plants showed good correlation between transgene expression and the level of sheath blight resistance among transformants. Functional complementation assays confirmed the effectiveness of the chitinase mediated resistance in all the transgenic TP309 plants with varying levels of enhanced resistance against R. solani. Therefore, the novel chitinase gene cloned and characterized in the present study from the QTL region of rice will be of significant use in molecular plant breeding program for developing sheath blight resistance in rice.

Specific and Sensitive Isothermal Electrochemical Biosensor for Plant Pathogen DNA Detection with Colloidal Gold Nanoparticles as Probes.

Developing quick and sensitive molecular diagnostics for plant pathogen detection is challenging. Herein, a nanoparticle based electrochemical biosensor was developed for rapid and sensitive detection of plant pathogen DNA on disposable screen-printed carbon electrodes. This 60 min assay relied on the rapid isothermal amplification of target pathogen DNA sequences by recombinase polymerase amplification (RPA) followed by gold nanoparticle-based electrochemical assessment with differential pulse voltammetry (DPV). Our method was 10,000 times more sensitive than conventional polymerase chain reaction (PCR)/gel electrophoresis and could readily identify P. syringae infected plant samples even before the disease symptoms were visible. On the basis of the speed, sensitivity, simplicity and portability of the approach, we believe the method has potential as a rapid disease management solution for applications in agriculture diagnostics.

Advanced DNA-Based Point-of-Care Diagnostic Methods for Plant Diseases Detection.

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.