Investigation of Asymptomatic Infection of Phellinus noxius in Herbaceous Plants.

The white-rot fungus Phellinus noxius is known to cause brown root rot disease (BRRD) in woody trees and shrubs. To understand the pathogenicity of P. noxius in herbaceous plants, we investigated 23 herbaceous weed and turfgrass species in 32 BRRD-infested sites in Taiwan and/or tested them by artificial inoculation. In the field survey, P. noxius was isolated from seven symptomless herbaceous species (i.e., Typhonium blumei, Paspalum conjugatum, Paspalum distichum, Oplismenus compositus, Bidens pilosa, Digitaria ciliaris, and Zoysia matrella). Potted plant inoculation assays suggested that P. noxius is able to infect Artemisia princeps, O. compositus, and Z. matrella but not Axonopus compressus, Eremochloa ophiuroides, Ophiopogon japonicus, or Cynodon dactylon. A. princeps plants wilted within 2 weeks postinoculation, but inoculated O. compositus and Z. matrella were asymptomatic, and P. noxius could be isolated from only inoculated sites. The colonization of P. noxius in the cortex and vascular cylinder of roots was visualized by paraffin sectioning and trypan blue staining of juvenile seedlings grown on water agar. To evaluate the effect of replantation for the remediation of BRRD-infested sites, P. noxius-inoculated wood strips were buried in soil with or without vegetation. After 4 weeks, P. noxius could be detected only in the bare soil group. For the control of BRRD, the herbaceous hosts should be removed around the diseased trees/stumps and non-host turfgrasses (e.g., A. compressus, E. ophiuroides, O. japonicus, or C. dactylon) planted to accelerate the degradation of P. noxius.

How to start your monocot CRISPR/Cas project: plasmid design, efficiency detection, and offspring analysis.

The breakthrough CRISPR (clustered regularly interspaced short palindromic repeat)/Cas9-mediated genome-editing technology has led to great progress in monocot research; however, several factors need to be considered for the efficient implementation of this technology. To generate genome-edited crops, single guide (sg)RNA and Cas9 DNA are delivered into plant cells and expressed, and the predicted position is targeted. Analyses of successful targeted mutations have revealed that the expression levels, expression timing, and variants of both sgRNA and Cas9 need to be sophisticatedly regulated; therefore, the promoters of these genes and the target site positions are the key factors for genome-editing efficiency. Currently, various vectors and online tools are available to aid sgRNA design. Furthermore, to reduce the sequence limitation of the protospacer adjacent motif (PAM) and for other purposes, many Cas protein variants and base editors can be used in plants. Before the stable transformation of a plant, the evaluation of vectors and target sites is therefore very important. Moreover, the delivery of Cas9-sgRNA ribonucleoproteins (RNPs) is one strategy that can be used to prevent transgene issues with the expression of sgRNA and Cas proteins. RNPs can be used to efficiently generate transgene-free genome-edited crops that can reduce transgene issues related to the generation of genetically modified organisms. In this review, we introduce new techniques for genome editing and identifying marker-free genome-edited mutants in monocot crops. Four topics are covered: the design and construction of plasmids for genome editing in monocots; alternatives to SpCas9; protoplasts and CRISPR; and screening for marker-free CRISPR/Cas9-induced mutants. We have aimed to encompass a full spectrum of information for genome editing in monocot crops.

Chlorophyll fluorescence analysis in diverse rice varieties reveals the positive correlation between the seedlings salt tolerance and photosynthetic efficiency.

BACKGROUND: Photosynthetic efficiency might be a key factor determining plant resistance to abiotic stresses. Plants can sense when growing conditions are not favorable and trigger an internal response at an early stage before showing external symptoms. When a high amount of salt enters the plant cell, the membrane system and function of thylakoids in chloroplasts could be destroyed and affect photosynthetic performance if the salt concentration is not regulated to optimal values. Oryza species have salt-tolerant and salt-sensitive genotypes; however, very few studies have investigated the genetic architecture responsible for photosynthetic efficiency under salinity stress in cultivated rice. RESULTS: We used an imaging-based chlorophyll fluorometer to monitor eight rice varieties that showed different salt tolerance levels for four consecutive days under control and salt conditions. An analysis of the changes in chlorophyll fluorescence parameters clearly showed the maximum quantum efficiency of PSII in sensitive varieties was significantly reduced after NaCl treatment when compared to tolerant varieties. A panel of 232 diverse rice accessions was then analyzed for chlorophyll fluorescence under salt conditions, the results showed that chlorophyll fluorescence parameters such as F0 and NPQ were higher in Japonica subspecies, ΦPSII of Indica varieties was higher than that in other subgroups, which suggested that the variation in photosynthetic efficiency was extensively regulated under salt treatment in diverse cultivated rice. Two significant regions on chromosome 5 were identified to associate with the fraction of open PSII centers (qL) and the minimum chlorophyll fluorescence (F0). These regions harbored genes related to senescence, chloroplast biogenesis and response to salt stress are of interest for future functional characterization to determine their roles in regulating photosynthesis. CONCLUSIONS: Rice plant is very sensitive to salinity stress, especially at young seedling stage. Our work identified the distribution pattern of chlorophyll fluorescence parameters in seedlings leaf and their correlations with salt tolerance level in a diverse gene pool. We also revealed the complexity of the genetic architecture regulating rice seedling photosynthetic performance under salinity stress, the germplasm analyzed in this study and the associated genetic information could be utilized in rice breeding program.

H2O2-Based Method for Rapid Detection of Transgene-Free Rice Plants from Segregating CRISPR/Cas9 Genome-Edited Progenies.

Genome-editing techniques such as CRISPR/Cas9 have been widely used in crop functional genomics and improvement. To efficiently deliver the guide RNA and Cas9, most studies still rely on Agrobacterium-mediated transformation, which involves a selection marker gene. However, several limiting factors may impede the efficiency of screening transgene-free genome-edited plants, including the time needed to produce each life cycle, the response to selection reagents, and the labor costs of PCR-based genotyping. To overcome these disadvantages, we developed a simple and high-throughput method based on visual detection of antibiotics-derived H2O2 to verify transgene-free genome-edited plants. In transgenic rice containing hygromycin phosphotransferase (HPT), H2O2 content did not change in the presence of hygromycin B (HyB). In contrast, in transgenic-free rice plants with 10-h HyB treatment, levels of H2O2 and malondialdehyde, indicators of oxidative stress, were elevated. Detection of H2O2 by 3,3'-diaminobenzidine (DAB) staining suggested that H2O2 could be a marker to efficiently distinguish transgenic and non-transgenic plants. Analysis of 24 segregating progenies of an HPT-containing rice plant by RT-PCR and DAB staining verified that DAB staining is a feasible method for detecting transformants and non-transformants. Transgene-free genome-edited plants were faithfully validated by both PCR and the H2O2-based method. Moreover, HyB induced overproduction of H2O2 in leaves of Arabidopsis, maize, tobacco, and tomato, which suggests the potential application of the DAB method for detecting transgenic events containing HPT in a wide range of plant species. Thus, visual detection of DAB provides a simple, cheap, and reliable way to efficiently identify transgene-free genome-edited and HPT-containing transgenic rice.

Response Regulators 9 and 10 Negatively Regulate Salinity Tolerance in Rice.

Cytokinins are involved in the regulation of many plant growth and development processes, and function in response to abiotic stress. Cytokinin signaling is similar to the prokaryotic two-component signaling systems and includes the transcriptional upregulation of type-A response regulators (RRs), which in turn act to inhibit cytokinin signal response via negative feedback. Cytokinin signaling consists of several gene families and only a handful full of genes is studied. In this study, we demonstrated the function of two highly identical type-A RR genes from rice, OsRR9 and OsRR10, which are induced by cytokinin and only OsRR10 repressed by salinity stress in rice. Loss-of-function mutations give rise to mutant genes, osrr9/osrr10, which have higher salinity tolerance than wild type rice seedlings. The transcriptomic analysis uncovered several ion transporter genes, which were upregulated in response to salt stress in the osrr9/osrr10 mutants relative to the wild type seedlings. These include high-affinity potassium transporters, such as OsHKT1;1, OsHKT1;3 and OsHKT2;1, which play an important role in sodium and potassium homeostasis. In addition, disruption of the genes OsRR9 and OsRR10 also affects the expression of multiple genes related to photosynthesis, transcription and phytohormone signaling. Taken together, these results suggest that the genes OsRR9 and OsRR10 function as negative regulators in response to salinity in rice.

Circadian oscillations of cytosolic free calcium regulate the Arabidopsis circadian clock.

In the last decade, the view of circadian oscillators has expanded from transcriptional feedback to incorporate post-transcriptional, post-translational, metabolic processes and ionic signalling. In plants and animals, there are circadian oscillations in the concentration of cytosolic free Ca2+ ([Ca2+]cyt), though their purpose has not been fully characterized. We investigated whether circadian oscillations of [Ca2+]cyt regulate the circadian oscillator of Arabidopsis thaliana. We report that in Arabidopsis, [Ca2+]cyt circadian oscillations can regulate circadian clock function through the Ca2+-dependent action of CALMODULIN-LIKE24 (CML24). Genetic analyses demonstrate a linkage between CML24 and the circadian oscillator, through pathways involving the circadian oscillator gene TIMING OF CAB2 EXPRESSION1 (TOC1).

Characterization of the cytokinin-responsive transcriptome in rice.

BACKGROUND: Cytokinin activates transcriptional cascades important for development and the responses to biotic and abiotic stresses. Most of what is known regarding cytokinin-regulated gene expression comes from studies of the dicotyledonous plant Arabidopsis thaliana. To expand the understanding of the cytokinin-regulated transcriptome, we employed RNA-Seq to analyze gene expression in response to cytokinin in roots and shoots of the monocotyledonous plant rice. RESULTS: We identified over 4,600 and approximately 2,400 genes differentially expressed in response to cytokinin in roots and shoots respectively. There were some similarities in the sets of cytokinin-regulated genes identified in rice and Arabidopsis, including an up-regulation of genes that act to reduce cytokinin function. Consistent with this, we found that the preferred DNA-binding motif of a rice type-B response regulator is similar to those from Arabidopsis. Analysis of the genes regulated by cytokinin in rice revealed a large number of transcription factors, receptor-like kinases, and genes involved in protein degradation, as well as genes involved in development and the response to biotic stress. Consistent with the over-representation of genes involved in biotic stress, there is a substantial overlap in the genes regulated by cytokinin and those differentially expressed in response to pathogen infection, suggesting that cytokinin plays an integral role in the transcriptional response to pathogens in rice, including the induction of a large number of WRKY transcription factors. CONCLUSIONS: These results begin to unravel the complex gene regulation after cytokinin perception in a crop of agricultural importance and provide insight into the processes and responses modulated by cytokinin in monocots.

Calmodulin-related CML24 interacts with ATG4b and affects autophagy progression in Arabidopsis.

Plants encounter environmental stress challenges that are distinct from those of other eukaryotes because of their relative immobility. Therefore, plants may have evolved distinct regulatory mechanisms for conserved cellular functions. Plants, like other eukaryotes, share aspects of both calcium- and calmodulin-based cellular signaling and the autophagic process of cellular renewal. Here, we report a novel function for an Arabidopsis calmodulin-related protein, CML24, and insight into ATG4-regulated autophagy. CML24 interacts with ATG4b in yeast two-hybrid, in vitro pull-down and transient tobacco cell transformation assays. Mutants with missense mutations in CML24 have aberrant ATG4 activity patterns in in vitro extract assays, altered ATG8 accumulation levels, an altered pattern of GFP-ATG8-decorated cellular structures, and altered recovery from darkness-induced starvation. Together, these results support the conclusion that CML24 affects autophagy progression through interactions with ATG4.

Characterization of genes involved in cytokinin signaling and metabolism from rice.

Two-component signaling elements play important roles in plants, including a central role in cytokinin signaling. We characterized two-component elements from the monocot rice (Oryza sativa) using several complementary approaches. Phylogenetic analysis reveals relatively simple orthologous relationships among the histidine kinases in rice and Arabidopsis (Arabidopsis thaliana). In contrast, the histidine-containing phosphotransfer proteins (OsHPs) and response regulators (OsRRs) display a higher degree of lineage-specific expansion. The intracellular localizations of several OsHPs and OsRRs were examined in rice and generally found to correspond to the localizations of their dicot counterparts. The functionality of rice type-B OsRRs was tested in Arabidopsis; one from a clade composed of both monocot and dicot type-B OsRRs complemented an Arabidopsis type-B response regulator mutant, but a type-B OsRR from a monocot-specific subfamily generally did not. The expression of genes encoding two-component elements and proteins involved in cytokinin biosynthesis and degradation was analyzed in rice roots and shoots and in response to phytohormones. Nearly all type-A OsRRs and OsHK4 were up-regulated in response to cytokinin, but other cytokinin signaling elements were not appreciably affected. Furthermore, multiple cytokinin oxidase (OsCKX) genes were up-regulated by cytokinin. Abscisic acid treatment decreased the expression of several genes involved in cytokinin biosynthesis and degradation. Auxin affected the expression of a few genes; brassinosteroid and gibberellin had only modest effects. Our results support a shared role for two-component elements in mediating cytokinin signaling in monocots and dicots and reveal how phytohormones can impact cytokinin function through modulating gene expression.

Nitric oxide accumulation in Arabidopsis is independent of NOA1 in the presence of sucrose.

Nitric oxide signals diverse responses in animals and plants. Whereas nitric oxide synthesis mechanisms in animals are well understood, how nitric oxide is synthesized and regulated in plants remains controversial. NOA1 is a circularly permuted GTPase that is important for chloroplast function and is implicated in nitric oxide synthesis. However, the reported consequences of a null mutation in NOA1 are inconsistent. Whereas some studies indicate that the noa1 mutant has severe reductions in nitric oxide accumulation, others report that nitric oxide levels are indistinguishable between noa1 and the wild type. Here, we identify a correlation between the reported ability of noa1 to accumulate nitric oxide with growth on sucrose-supplemented media. We report that noa1 accumulates both basal and salicylic acid-induced nitric oxide only when grown on media containing sucrose. In contrast, nitric oxide accumulation in wild type is largely insensitive to sucrose supplementation. When grown in the absence of sucrose, noa1 has low fumarate, pale green leaves, slow growth and reduced chlorophyll content. These phenotypes are consistent with a defect in chloroplast-derived photosynthate production and are largely rescued by sucrose supplementation. We conclude that NOA1 has a primary role in chloroplast function and that its effects on the accumulation of nitric oxide are likely to be indirect.

Developmental phytotoxicity of metal oxide nanoparticles to Arabidopsis thaliana.

Phytotoxicity is an important consideration to understand the potential environmental impacts of manufactured nanomaterials. Here, we report on the effects of four metal oxide nanoparticles, aluminum oxide (nAl(2)O(3)), silicon dioxide (nSiO(2)), magnetite (nFe(3)O(4)), and zinc oxide (nZnO), on the development of Arabidopsis thaliana (Mouse-ear cress). Three toxicity indicators (seed germination, root elongation, and number of leaves) were quantified following exposure to each nanoparticle at three concentrations: 400, 2,000, and 4,000 mg/L. Among these particles, nZnO was most phytotoxic, followed by nFe(3)O(4), nSiO(2), and nAl(2)O(3), which was not toxic. Consequently, nZnO was further studied to discern the importance of particle size and zinc dissolution as toxicity determinants. Soluble zinc concentrations in nanoparticle suspensions were 33-fold lower than the minimum inhibitory concentration of dissolved zinc salt (ZnCl(2)), indicating that zinc dissolution could not solely account for the observed toxicity. Inhibition of seed germination by ZnO depended on particle size, with nanoparticles exerting higher toxicity than larger (micron-sized) particles at equivalent concentrations. Overall, this study shows that direct exposure to nanoparticles significantly contributed to phytotoxicity and underscores the need for eco-responsible disposal of wastes and sludge containing metal oxide nanoparticles.

Innate immunity signaling: cytosolic Ca2+ elevation is linked to downstream nitric oxide generation through the action of calmodulin or a calmodulin-like protein.

Ca(2+) rise and nitric oxide (NO) generation are essential early steps in plant innate immunity and initiate the hypersensitive response (HR) to avirulent pathogens. Previous work from this laboratory has demonstrated that a loss-of-function mutation of an Arabidopsis (Arabidopsis thaliana) plasma membrane Ca(2+)-permeable inwardly conducting ion channel impairs HR and that this phenotype could be rescued by the application of a NO donor. At present, the mechanism linking cytosolic Ca(2+) rise to NO generation during pathogen response signaling in plants is still unclear. Animal nitric oxide synthase (NOS) activation is Ca(2+)/calmodulin (CaM) dependent. Here, we present biochemical and genetic evidence consistent with a similar regulatory mechanism in plants: a pathogen-induced Ca(2+) signal leads to CaM and/or a CaM-like protein (CML) activation of NOS. In wild-type Arabidopsis plants, the use of a CaM antagonist prevents NO generation and the HR. Application of a CaM antagonist does not prevent pathogen-induced cytosolic Ca(2+) elevation, excluding the possibility of CaM acting upstream from Ca(2+). The CaM antagonist and Ca(2+) chelation abolish NO generation in wild-type Arabidopsis leaf protein extracts as well, suggesting that plant NOS activity is Ca(2+)/CaM dependent in vitro. The CaM-like protein CML24 has been previously associated with NO-related phenotypes in Arabidopsis. Here, we find that innate immune response phenotypes (HR and [avirulent] pathogen-induced NO elevation in leaves) are inhibited in loss-of-function cml24-4 mutant plants. Pathogen-associated molecular pattern-mediated NO generation in cells of cml24-4 mutants is impaired as well. Our work suggests that the initial pathogen recognition signal of Ca(2+) influx into the cytosol activates CaM and/or a CML, which then acts to induce downstream NO synthesis as intermediary steps in a pathogen perception signaling cascade, leading to innate immune responses, including the HR.

Expression of ASCORBATE PEROXIDASE 8 in roots of rice (Oryza sativa L.) seedlings in response to NaCl.

Reactive oxygen species are thought to play an important role in NaCl stress. Therefore, the expression patterns of the gene family encoding the H(2)O(2)-scavenging enzyme ascorbate peroxidase (APx; EC1.11.1.11) were analysed in roots of etiolated rice (Oryza sativa L.) seedlings in response to NaCl stress. Applying semi-quantitative RT-PCR, the mRNA levels were quantified for two cytosolic (OsAPx1 and OsAPx2), two peroxisomal (OsAPx3 and OsAPx4), and four chloroplastic (OsAPx5, OsAPx6, OsAPx7, and OsAPx8) isoforms identified in the rice genome. NaCl at 150 mM and 200 mM increased the expression of OsAPx8 and the activities of APx, but had no effect on the expression of OsAPx1, OsAPx2, OsAPx3, OsAPx4, OsAPx5, OsAPx6, and OsAPx7 in rice roots. However, NaCl at 300 mM up-regulated OsAPx8 expression, increased APx activity, and down-regulated OsAPx7 expression, but had no effect on the expression of OsAPx1, OsAPx2, OsAPx3, OsAPx4, OsAPx5, and OsAPx6. The accumulation of abscisic acid (ABA) in response to NaCl was observed in rice roots. Exogenously applied ABA also specifically enhanced the expression of OsAPx8 in rice roots. The accumulation of ABA in rice roots in response to NaCl was inhibited by fluridone (Flu), an inhibitor of carotenoid biosynthesis. Flu treatment also suppressed NaCl-enhanced OsAPx8 expression and APx activity. The effect of Flu on the expression of OsAPx8 and increase in APx activity was reversed by the application of ABA. It appears that NaCl-enhanced expression of OsAPx8 in rice roots is mediated through an accumulation of ABA. Evidence is provided to show that Na(+) but not Cl(-) is required for enhancing OsAPx8 expression, APx activity, and ABA accumulation in rice roots treated with NaCl. H(2)O(2) treatment resulted in an enhancement of OsAPx8 induction but no accumulation of ABA. Diphenylene iodonium treatment, which is known to inhibit NaCl-induced accumulation of H(2)O(2) in rice roots, did not suppress OsAPx8 induction and ABA accumulation by NaCl. It appears that H(2)O(2) is not involved in the regulation of NaCl-induced OsAPx8 expression in rice roots.

Arabidopsis potential calcium sensors regulate nitric oxide levels and the transition to flowering.

In plants, flowering is a critical developmental transition orchestrated by four regulatory pathways. Distinct alleles encoding mutant forms of the Arabidopsis potential calcium sensor CML24 cause alterations in flowering time. CML24 can act as a switch in the response to day length perception; loss-of-function cml24 mutants are late flowering under long days, whereas apparent gain of CML24 function results in early flowering. CML24 function is required for proper CONSTANS (CO) expression; components upstream of CO in the photoperiod pathway are largely unaffected in the cml24 mutants. In conjunction with CML23, a related calmodulin-like protein, CML24 also inhibits FLOWERING LOCUS C (FLC) expression and therefore impacts the autonomous regulatory pathway of the transition to flowering. Nitric oxide (NO) levels are elevated in cml23/cml24 double mutants and are largely responsible for FLC transcript accumulation. Therefore, CML23 and CML24 are potential calcium sensors that have partially overlapping function that may act to transduce calcium signals to regulate NO accumulation. In turn, NO levels influence the transition to flowering through both the photoperiod and autonomous regulatory pathways.

Handling calcium signaling: Arabidopsis CaMs and CMLs.

The Arabidopsis genome harbors seven calmodulin (CAM) and 50 CAM-like (CML) genes that encode potential calcium sensors. The CAMs encode only four protein isoforms. Selective pressure to maintain multiple CAMs indicates nonredundancy. Sequence divergence, even in the EF hand calcium-binding motif, exists among the CMLs and, therefore, divergent functions are likely to have evolved. Expression data recently available from Massively Parallel Signature Sequencing and Genevestigator compilation of microarrays are reviewed. The seven Arabidopsis CAMs are highly and relatively uniformly expressed. Differential expression is evident among the distinct CMLs over developmental stages, in various organs and in response to many different stimuli. In spite of the potential importance in mediating plant calcium signaling, the physiological functions of the Arabidopsis CaMs and CMLs remain largely unknown.

Expression of ascorbate peroxidase and glutathione reductase in roots of rice seedlings in response to NaCl and H2O2.

The accumulation of H2O2 by NaCl was observed in the roots of rice seedlings. Treatment with NaCl caused an increase in the activities of ascorbate peroxidase (APX) and glutathione reductase (GR) and the expression of OsAPX and OsGR in rice roots. Exogenously applied H2O2 also enhanced the activities of APX and GR and the expression of OsAPX and OsGR in rice roots. The accumulation of H2O2 in rice roots in response to NaCl was inhibited by the NADPH oxidase inhibitors, diphenyleneiodonium chloride (DPI) and imidazole (IMD). However, DPI, IMD, and dimethylthiourea, a H2O2 trap, did not reduce NaCl-enhanced activities of APX and GR and expression of OsAPX and OsGR. It appears that H2O2 is not involved in the regulation of NaCl-induced APX and GR activities and OsAPX and OsGR expression in rice roots.