Biology subjects

Char, S. N.

Publications and source records attributed to Char, S. N..

10 recordsLinked to original sources

bioRxiv2024

Small DNA elements that act as both insulators and silencers in plants

Insulators are cis-regulatory elements that separate transcriptional units, whereas silencers are elements that repress transcription regardless of their position. In plants, these elements remain largely uncharacterized. Here, we use the massively parallel reporter assay Plant STARR-seq with short fragments of eight large insulators to identify more than 100 fragments that block enhancer activity. The short fragments can be combined to generate more powerful insulators that abolish the capacity of the strong viral 35S enhancer to activate the 35S minimal promoter. Unexpectedly, when tested upstream of weak enhancers, these fragments act as silencers and repress transcription. Thus, these elements are capable of both insulating or repressing transcription dependent upon regulatory context. We validate our findings in stable transgenic Arabidopsis, maize, and rice plants. The short elements identified here should be useful building blocks for plant biotechnology efforts.

plant biology↗

bioRxiv2024

Activation of two noncanonical R proteins by an insect effector confers plant immunity to aphid infestation

Molecular characterization of resistance genes is crucial for efficiently understanding and fortifying plant immunity against insect herbivores. Here we report that RMES1A and RMES1B proteins confer resistance to the sorghum aphid Melanaphis sorghi when activated by an insect effector MsEF1. Map-based cloning plus genetic analysis of knockout mutants confirm that RMES1A and RMES1B are both required for aphid resistance. Upon aphid attack, RMES1A and RMES1B expression is elevated in the sclerenchyma cells and vascular bundles of leaves; the two proteins interact with MsEF1 in the exocysts, thus upregulating key defense processes such as reactive oxygen species burst. Structural modeling predicts that RMES1A and RMES1B each carry an ATP binding site and two leucine-rich-repeat domains but lack coiled-coil or Toll/Interleukin-1 receptor/resistance domain, thus likely representing a new type of resistance controlling proteins in plants. Our work reveals new genes and mechanisms for further deciphering and improving plant immunity to insect pests.

genetics↗

bioRxiv2023

Efficient protein tagging and cis-regulatory element engineering via precise and directional oligonucleotide-based targeted insertion in plants

Efficient and precise targeted insertion holds great promise but remains challenging in plant genome editing. An efficient NHEJ-mediated targeted insertion method was recently developed by combining CRISPR-Cas9 with phosphorothioate modified double-stranded oligodeoxynucleotides (dsODNs). Yet this approach often led to imprecise insertions with no control over the insertion direction. In this study, we first quantitatively compared the impact of the chemical protection on efficiency of targeted insertion. With the observation that CRISPR-SpCas9 could frequently induce staggered cleavages with 5' 1-nucleotide overhangs, we then evaluated the impact of the donor end structures on the direction and preciseness of targeted insertions. Our study demonstrated that the chemically protected dsODNs with 5' 1-nt overhangs could significantly improve the precision and direction control of target insertions in all tested CRIPSR targeting sites. Lastly, we applied this method to endogenous gene tagging in Setaria viridis, and cis-regulatory element engineering for disease resistance in rice. Two distinct TAL effector binding elements were directionally inserted into the promoter region of a recessive rice bacterial blight resistance gene at up to 24.4% efficiency. The resulting rice lines with heritable insertions exhibited strong resistance to the infection of Xanthomonas oryzae pv. oryzae pathogen in an inducible and strain-specific manner.

plant biology↗

bioRxiv2022

Genome editing of an African elite rice variety confers resistance against endemic and emerging Xanthomonas oryzae pv. oryzae strains

Bacterial leaf blight (BB) of rice, caused by Xanthomonas oryzae pv. oryzae (Xoo), threatens global food security and the livelihood of small-scale rice producers. Analyses of Xoo collections from Asia, Africa and the Americas demonstrated surprising continental segregation, despite robust global rice trade. Here, we report unprecedented BB outbreaks in Tanzania. The causative strains, unlike endemic Xoo, carry Asian-type TAL effectors targeting the sucrose transporter SWEET11a and suppressing Xa1. Phylogenomics clustered these strains with Xoo strains from China. African rice varieties do not carry suitable resistance genes. To protect African rice production against this emerging threat, we developed a hybrid CRISPR-Cas9/Cpf1 system to edit six TALe-binding elements in three SWEET promoters of the East African elite variety Komboka. The edited lines show broad-spectrum resistance against Asian and African strains of Xoo, including strains recently discovered in Tanzania. This strategy could help to protect global rice crops from BB pandemics.

plant biology↗

bioRxiv2022

A dolabralexin-deficient mutant provides insight into specialized diterpenoid metabolism in maize (Zea mays).

Two major groups of maize (Zea mays) specialized metabolites, termed kauralexins and dolabralexins, serve as known or predicted diterpenoid defenses against pathogens, herbivores, and other environmental stressors. To consider physiological roles of the recently discovered dolabralexin pathway, we examined dolabralexin structural diversity, tissue-specificity, and stress-elicited production in a defined biosynthetic pathway mutant. Metabolomics analyses support a larger number of dolabralexin pathway products than previously known. We identified dolabradienol as a previously undetected pathway metabolite and characterized its enzymatic production. Transcript and metabolite profiling showed that dolabralexin biosynthesis and accumulation predominantly occurs in primary roots and shows quantitative variation across genetically diverse inbred lines. Generation and analysis of CRISPR-Cas9-derived loss-of- function Kaurene Synthase-Like 4 (Zmksl4) mutants demonstrated dolabralexin production deficiency, thus supporting ZmKSL4 as the diterpene synthase responsible for the conversion of geranylgeranyl pyrophosphate precursors into dolabradiene and downstream pathway products. Zmksl4 mutants further display altered root-to-shoot ratios and root architecture in response to water deficit, consistent with an interactive role of maize dolabralexins in plant vigor during abiotic stress.

plant biology↗

bioRxiv2022

Polarly localized WPR proteins interact with PAN receptors and the actin cytoskeleton during maize stomatal development

Polarization of cells prior to asymmetric cell division is crucial for correct cell divisions, cell fate and tissue patterning. In maize stomatal development, polarization of subsidiary mother cells prior to asymmetric division is controlled by the BRK-PAN-ROP pathway. Two catalytically inactive receptor-like kinases, PAN2 and PAN1, are required for correct division plane positioning. Proteins in the BRK-PAN-ROP pathway are polarized in subsidiary mother cells, with the polarization of each protein dependent on the previous one. As most of the known proteins in this pathway do not physically interact, possible interactors that might participate in the pathway are yet to be described. We identified WPR proteins as new players during subsidiary mother cell polarization. WPRs physically interact with PAN receptors, and polarly accumulate in subsidiary mother cells. The polarized localization of WPR proteins depends on PAN2 but not PAN1. CRISPR-Cas9- induced mutations result in division plane defects in subsidiary mother cells, and ectopic expression of WPR-RFP results in stomatal defects and alterations to the actin cytoskeleton. We show certain WPR proteins directly interact with F-actin through their N-terminus. Our data implicate WPR proteins as potentially regulating actin filaments, which providing insight into their molecular function. Together, these results demonstrate that WPR proteins are important for cell polarization. One-sentence summaryFour related proteins, identified via their physical interaction with the receptor PAN2, are polarly localized prior to asymmetric division in stomatal lineage cells, and interact with F-actin.

plant biology↗

bioRxiv2021

OsSWEET11b, a sixth leaf blight susceptibility gene involved in sugar transport-dependent male fertility

SWEETs play important roles in intercellular sugar transport. Induction of SWEET sugar transporters by transcription activator-like effectors (TALe) of Xanthomonas ssp. is a key factor for bacterial leaf blight (BLB) infection of rice, cassava and cotton. Here, we identified the so far unknown OsSWEET11b with roles in male fertility and BLB susceptibility in rice. While single ossweet11a or b mutants were fertile, double mutants were sterile. Since clade III SWEETs can transport gibberellin (GA), a key hormone for rice spikelet fertility, sterility and BLB susceptibility might be explained by GA transport deficiencies. However, in contrast to the Arabidopsis homologs, OsSWEET11b did not mediate detectable GA transport. Fertility and susceptibility must therefore depend on SWEET11b-mediated sucrose transport. Ectopic induction of OsSWEET11b by designer TALe enables TALe-free Xanthomonas oryzae pv. oryzae (Xoo) to cause disease, identifying OsSWEET11b as a BLB susceptibility gene and demonstrating that the induction of host sucrose uniporter activity is key to virulence of Xoo. Notably, only three of now six clade III SWEETs are targeted by known Xoo strains from Asia and Africa. The identification of OsSWEET11b has relevance in the context of fertility and for protecting rice against emerging Xoo strains that evolve TALes to exploit OsSWEET11b.

plant biology↗

bioRxiv2020

Genetic elucidation of complex biochemical traits mediating maize innate immunity

Specialized metabolites constitute key layers of immunity underlying crop resistance; however, challenges in resolving complex pathways limit our understanding of their functions and applications. In maize (Zea mays) the inducible accumulation of acidic terpenoids is increasingly considered as a defense regulating disease resistance. To understand maize antibiotic biosynthesis, we integrated association mapping, pan-genome multi-omic correlations, enzyme structure-function studies, and targeted mutagenesis. We now define ten genes in three zealexin (Zx) gene clusters comprised of four sesquiterpene synthases and six cytochrome P450s that collectively drive the production of diverse antibiotic cocktails. Quadruple mutants blocked in the production of {beta}-macrocarpene exhibit a broad-spectrum loss of disease resistance. Genetic redundancies ensuring pathway resiliency to single null mutations are combined with enzyme substrate-promiscuity creating a biosynthetic hourglass pathway utilizing diverse substrates and in vivo combinatorial chemistry to yield complex antibiotic blends. The elucidated genetic basis of biochemical phenotypes underlying disease resistance demonstrates a predominant maize defense pathway and informs innovative strategies for transferring chemical immunity between crops.

plant biology↗

bioRxiv2019

The maize heterotrimeric G-protein β subunit controls shoot meristem development and immune responses.

Heterotrimeric G-proteins are important transducers of receptor signaling, functioning in plants with CLAVATA receptors in control of shoot meristem size, and with pathogen associated molecular pattern (PAMP) receptors in basal immunity. However, whether specific members of the heterotrimeric complex potentiate crosstalk between development and defense, and the extent to which these functions are conserved across species, has not been addressed. Here we used CRISPR/Cas9 to knockout the maize G{beta} subunit gene, and found that the mutants were lethal, differing from Arabidopsis, where homologous mutants have normal growth and fertility. We show that lethality is not caused by a specific developmental arrest, but by autoimmunity. We used a genetic diversity screen to suppress the lethal g{beta} phenotype, and also identified a new maize G{beta} allele with weak autoimmune responses but strong development phenotypes. Using these tools, we show that G{beta} controls meristem size in maize, acting epistatically with G, suggesting that G{beta} and G function in a common signaling complex. Furthermore, we used an association study to show that natural variation in G{beta} influences maize kernel row number, an important agronomic trait. Our results demonstrate the dual role of G{beta} in immunity and development in a cereal crop, and suggest that it functions in crosstalk between these competing signaling networks. Therefore, modification of G{beta} has the potential to optimize the tradeoff between growth and defense signaling to improve agronomic production.\n\nSignificanceCereal crops, such as maize provide our major food and feed. Crop productivity has been significantly improved by selection of favorable architecture and development alleles, however crops are constantly under attack from pathogens, which severely limits yield due to a defense-growth tradeoff. Therefore, it is critical to identify key signaling regulators that control both developmental and immune signaling, to provide basic knowledge to maximize productivity. This work shows that the maize G protein {beta} subunit regulates both meristem development and immune signaling, and suggests that manipulation of this gene has the potential to optimize the tradeoff between yield and disease resistance to improve crop yields.

plant biology↗

bioRxiv2017

Impaired phloem loading in genome-edited triple knock-out mutants of SWEET13 sucrose transporters

Crop yield depends on efficient allocation of sucrose from leaves to seeds. In Arabidopsis, phloem loading is mediated by a combination of SWEET sucrose effluxers and subsequent uptake by SUT1/SUC2 sucrose/H+ symporters. ZmSUT1 is essential for carbon allocation in maize, but the relative contribution to apoplasmic phloem loading and retrieval of sucrose leaking from the translocation path is not known. We therefor tested whether SWEETs are important for phloem loading in maize.\n\nHere we identified three leaf-expressed SWEET sucrose transporters as key components of apoplasmic phloem loading in Zea mays L. Notably, ZmSWEET13 paralogs (a, b, c) are among the highest expressed genes in the leaf vasculature. Genome-edited triple knock-out mutants are severely stunted. Photosynthesis of mutants was impaired and leaves accumulated starch and soluble sugars. RNA-seq revealed profound transcriptional deregulation of genes associated with the photosynthetic apparatus and carbohydrate metabolism. GWAS analyses may indicate that variability in ZmSWEET13s is correlated with agronomical traits, specifically flowering time and leaf angle.\n\nThis work provides support for cooperation of three ZmSWEET13s with ZmSUT1 in phloem loading in Zea mays L. Our study highlights these three ZmSWEET13 sucrose transporters as possible candidates for the engineering of crop yield.\n\nOne Sentence Summary: Three SWEET sucrose transporter paralogs are necessary for phloem loading in maize.

plant biology↗