Biology subjects

Rast-Somssich, M. I.

Publications and source records attributed to Rast-Somssich, M. I..

4 recordsLinked to original sources

bioRxiv2025

From lab to field: analyses of genome-edited bacterial blight resistant rice

Bacterial blight (BB) of rice, caused by Xanthomonas oryzae pv. oryzae (Xoo) is one of the major drivers of yield losses in Africa and Asia. Xoo secretes TAL-effectors (TALe) that induce host SWEET sucrose uniporter by binding to the effector binding element (EBE) of SWEET promoters, likely required for Xoo reproduction and virulence. We had multiplex edited the EBEs of three SWEET genes to prevent TALe binding, producing genome-edited (GEd) rice mega-varieties (IR64, Ciherang-Sub1 for Asia, and Komboka for Africa) that were resistant to a wide spectrum of Xoo strains. Here, we report comprehensive analyses of the GEd lines, including evaluation of agronomic performance in multi-location multi-season experimental field plots under different fertilization regimes, and tests for the presence/absence of foreign DNA/transgene in the offspring of GEd lines (IR64-BC1T6, Ciherang-Sub1-BC1T5, Komboka-T3). Various strategies were evaluated, including herbicide tolerance, PCR, DNA gel blotting, whole genome sequencing (WGS), and specific tests stipulated by country-specific biosafety guidelines. Different WGS technologies were evaluated and also used to identify heritability of the edits, single nucleotide polymorphisms (SNPs), and insertions/deletions (indels) that might have resulted from somaclonal variation and potential GE-induced off-target mutations. Complete genome reference sequences for the parental lines IR64, Ciherang-Sub1, and Komboka are provided. In the field experiments, the GEd lines did not show performance defects. Together, the results indicate that select GE lines do not contain foreign DNA or transgene fragments and fulfill the requirements for treatment equivalent to classical breeding lines in countries such as India and Kenya.

plant biology↗

bioRxiv2022

pGG-PIP: A GreenGate (GG) entry vector collection with Plant Immune system Promoters (PIP)

The regulatory sequences controlling the expression of a gene (i.e., the promoter) are essential to properly understand a genes function. From their use in mutant complementation assays, to studying their responsiveness to different stimuli via transcriptional reporter lines or using them as proxy for the activation of certain pathways, assays using promoter sequences are valuable tools for insight into the genetic architecture underlying plant life. The GreenGate (GG) system is a plant-specific variant of the Golden Gate assembly method, a modular cloning system that allows the hierarchical assembly of individual donor DNA fragments into one expression clone via a single reaction step. Here, we present a collection of 75 GG entry vectors carrying putative regulatory sequences for Arabidopsis thaliana genes involved in many different pathways of the plant immune system, designated Plant Immune system Promoters (PIP). This pGG-PIP entry vector set enables the rapid assembly of expression vectors to be used for transcriptional reporters of plant immune system components, mutant complementation assays when coupled with coding sequences, mis-expression experiments for genes of interest, or the targeted use of CRISPR/Cas9 genome editing. We used pGG-PIP vectors to create fluorescent transcriptional reporters in A. thaliana and demonstrated the potential of these reporters to image the responsiveness of specific plant immunity genes to infection and colonization by the fungal pathogen Fusarium oxysporum. Using the PLANT ELICITOR PEPTIDE (PEP) pathway as an example, we show that several components of this pathway are locally activated in response to colonization by the fungus.

plant biology↗

bioRxiv2022

Spatially distinct phytohormone responses of individual Arabidopsis thaliana root cells to infection and colonization by Fusarium oxysporum

Jasmonic acid (JA), ethylene (ET) and salicylic acid (SA) are the three major phytohormones coordinating a plants defense response to pathogenic attack. While JA and ET are assumed to primarily control the defense against necrotrophic pathogens, SA-induced defense responses target mainly biotrophic microbes, and can include drastic measures such as programmed cell death as part of the plants hypersensitive response (HR). Fusarium oxysporum is a hemibiotrophic fungal pathogen of several plant species, including many important food crops, and the model plant species Arabidopsis thaliana. Colonization of the plants root vascular tissue by the fungus eventually results in wilting and plant death. A general role for JA, ET and SA in combating infection and colonization of the plant by F. oxysporum has been demonstrated, but their distinct roles and modes of action have so far not been described. Here, using high resolution microscopy with fluorescent marker lines of A. thaliana roots infected with F. oxysporum we show that SA acts spatially separate from JA, in a distinct set of root cells immediately neighboring the fungal colonization site. There, SA induces HR to stop the spread of colonization. JA acts in a different, but also clearly defined set of cells, slightly removed from the colonization site, where it initiates a defense response to actively resist the invader. ET is activated in a stretch of cells that covers both, the cells with activated SA and JA signaling, and may be involved in creating these two distinct zones. These results show how the three phytohormones act together, but spatially and functionally separate from each other, to fight this hemibiotrophic pathogen. Such a high-resolution analysis to resolve the plants immune response to pathogenic infection on an individual cell level and in intact tissue has so far been lacking. Graphical AbstractO_LIColonization of the A. thaliana root tip by F. oxysporum strain Fo5176 leads to immediate cell death of the colonized and surrounding tissue. C_LIO_LIAs the colonization front progresses through the vasculature, the cell death front moves along with it through not only the vasculature, but also the surrounding tissues. C_LIO_LIWRKY70 positively regulates salicylic acid (SA) biosynthesis in cells immediately adjacent to the colonized tissue, inducing a hypersensitive response (HR), thereby killing off the cells deemed lost to the intruder, establishing the cell death front. C_LIO_LISlightly further removed from the HR zone, WRKY11 induces jasmonate (JA) biosynthesis in cells of the vasculature to launch a defense response aimed at actively repelling the fungus. C_LI O_FIG O_LINKSMALLFIG WIDTH=122 HEIGHT=200 SRC="FIGDIR/small/521292v2_ufig1.gif" ALT="Figure 1"> View larger version (50K): org.highwire.dtl.DTLVardef@1bd280forg.highwire.dtl.DTLVardef@191052aorg.highwire.dtl.DTLVardef@1ea43acorg.highwire.dtl.DTLVardef@100ecdb_HPS_FORMAT_FIGEXP M_FIG C_FIG

plant biology↗

bioRxiv2020

Gamete-specific expression of TALE class HD genes activates the diploid sporophyte program in Marchantia polymorpha

Eukaryotic life cycles alternate between haploid and diploid phases and in phylogenetically diverse unicellular eukaryotes, expression of paralogous homeodomain genes in the two gametes directs the haploid-to-diploid transition. In the unicellular Chlorophyte alga Chlamydomonas KNOX and BELL TALE-homeodomain genes mediate the transition. Here we demonstrate that in the liverwort Marchantia polymorpha paternal (sperm) expression three of the five phylogenetically diverse BELL genes, MpBELL234, and maternal (egg) expression of MpKNOX1 mediate the haploid-to-diploid transition. Loss-of-function alleles of either result in zygotic or early embryonic arrest. In land plants both the haploid gametophyte and diploid sporophyte are complex multicellular organisms. Expression of MpKNOX1 and two other paralogs, MpBELL1 and MpKNOX2, during sporophyte development is consistent with a later role in patterning the sporophyte. These results indicate that the ancestral mechanism to activate diploid gene expression was retained in early diverging land plants and subsequently co-opted during evolution of the diploid sporophyte body.

developmental biology↗