Jennette Marie Codjoe

SUMMARY: Pennycress ( Thlaspi arvense ) is a winter oilseed domesticated recently to be incorporated as an intermediate crop between the existing cropping systems of the US Midwest. We show that a natural accession of pennycress, 2032, is more susceptible to the necrotrophic fungal pathogens Sclerotinia sclerotiorum and Alternaria japonica than the reference pennycress accession MN106. A previously identified marker associated with early flowering and maturity in pennycress was found to be present in a gene homologous to Arabidopsis Jumonji 14 ( JMJ14 ). It has been reported that AtJMJ14 promotes disease resistance and represses flowering, and greenhouse studies of breeding populations suggested that this was also the case in pennycress. Plants with the 2032 TaJMJ14 allele were more susceptible to fungi and flowered early. CRISPR‐Cas9 editing was used to generate additional TaJMJ14 alleles. A 9‐base pair deletion in the sixth exon of TaJMJ14 showed trends of early flowering and S. sclerotiorum susceptibility, whereas a complete loss‐of‐function allele led to infertility. We further investigated the transcriptomes of MN106 and 2032 plants in the early stages of S. sclerotiorum and A. japonica infection to identify potential resistance and susceptibility genes. Differences in the expression of pathogen‐associated molecular pattern‐triggered immunity‐associated genes led us to discover that 2032 plants have defects in elicitor‐triggered oxidative bursts. The transcriptional responses unique to each accession lay a foundation for future gene editing and breeding approaches to keep the beneficial early flowering phenotype conferred by 2032 but uncouple it from disease susceptibility.

Field Pennycress ( Thlaspi arvense ) is gaining attention in the US Midwest as a potential oilseed cover crop for their corn-soybean systems. Field research with breeding trials have shown that pennycress is susceptible to major fungal diseases affecting Brassica crops. In this study, we identified two pathogens: Alternaria japonica and Sclerotinia sclerotiorum which cause Alternaria black spot and Sclerotinia white mold diseases, respectively. Both fungi infect pennycress leaves and pods, with S. sclerotiorum also able to infect stems of the two pennycress accessions tested. We found accession 2032 to be more susceptible than MN106. Traditional visual methods to estimate disease severity could not capture the differential progression of Alternaria black spot in the two pennycress accessions. To address this, we developed a cost-effective DNA-based qPCR-based assay that can detect differential growth of both A. japonica and S. sclerotiorum on leaves and pods of two pennycress accessions. This method also provided more precise quantification of A. japonica and S. sclerotiorum at early infection stages than was possible visually. This assay could be helpful in evaluating various pennycress cultivars and other crops affected by these pathogens.

Pennycress ( Thlaspi arvense ) is a winter oilseed domesticated recently to be incorporated as an intermediate crop between the existing cropping systems of the US Midwest. We show that a natural accession of pennycress, 2032, is more susceptible to the necrotrophic fungal pathogens Sclerotinia sclerotiorum and Alternaria japonica than the reference pennycress accession MN106. A previously identified marker associated with early flowering and maturity in pennycress was found to be present in a gene homologous to Arabidopsis Jumonji 14 (JMJ14). It has been reported that AtJMJ14 promotes disease resistance and represses flowering, and greenhouse studies of breeding populations confirmed this phenomenon in pennycress. Plants with the 2032 TaJMJ14 allele were more susceptible to fungi and flowered early. CRISPR-Cas9 editing was used to generate additional TaJMJ14 alleles. A 9-base pair deletion in the 6 ^th exon of TaJMJ14 showed trends of early flowering and S. sclerotiorum susceptibility, whereas a complete loss-of-function allele led to infertility. We further investigated the transcriptomes of MN106 and 2032 plants in the early stages of S. sclerotiorum and A. japonica infection to identify potential resistance and susceptibility genes. Differences in the expression of pathogen-associated molecular pattern-triggered immunity (PTI)-associated genes led us to discover that 2032 plants have defects in elicitor-triggered oxidative bursts. The transcriptional responses unique to each accession lay a foundation for future gene-editing and breeding approaches to keep the beneficial early flowering phenotype conferred by 2032 but uncouple it from disease susceptibility.

White mold disease caused by a necrotrophic ascomycete pathogen Sclerotinia sclerotiorum results in serious economic losses of soybean yield in the USA. Lack of effective genetic resistance to this disease in soybean germplasm and increasing pathogen resistance to fungicides makes white mold difficult to manage. Small cysteine-rich antifungal peptides with multi-faceted modes of action possess potential for development as sustainable spray-on bio-fungicides. We have previously reported that GMA4CG_V6 peptide, a 17-amino acid variant of the MtDef4 defensin-derived peptide GMA4CG containing the active γ-core motif, exhibits potent antifungal activity against the gray mold fungal pathogen Botrytis cinerea in vitro and in planta. GMA4CG_V6 exhibited antifungal activity against an aggressive field isolate of S. sclerotiorum 555 in vitro with an MIC value of 24 µM. At this concentration, internalization of this peptide into fungal cells occurred prior to discernible membrane permeabilization. GMA4CG_V6 markedly reduced white mold disease symptoms when applied to detached soybean leaves, pods, and stems. Its spray application on soybean plants provided robust control of this disease. GMA4CG_V6 at sub-lethal concentrations reduced sclerotia production. It was also non-phytotoxic to soybean plants. Our results demonstrate that GMA4CG_V6 peptide has potential for development as a bio-fungicide for white mold control in soybean.

Intrinsically disordered protein regions (IDRs) are highly dynamic sequences that rapidly sample a collection of conformations over time. In the past several decades, IDRs have emerged as a major component of many proteomes, comprising ~30% of all eukaryotic protein sequences. Proteins with IDRs function in a wide range of biological pathways and are notably enriched in signaling cascades that respond to environmental stresses. Here, we identify and characterize intrinsic disorder in the soluble cytoplasmic N‐terminal domains of MSL8, MSL9, and MSL10, three members of the MscS‐like (MSL) family of mechanosensitive ion channels. In plants, MSL channels are proposed to mediate cell and organelle osmotic homeostasis. Bioinformatic tools unanimously predicted that the cytosolic N‐termini of MSL channels are intrinsically disordered. We examined the N‐terminus of MSL10 (MSL10^N) as an exemplar of these IDRs and circular dichroism spectroscopy confirms its disorder. MSL10^N adopted a predominately helical structure when exposed to the helix‐inducing compound trifluoroethanol (TFE). Furthermore, in the presence of molecular crowding agents, MSL10^N underwent structural changes and exhibited alterations to its homotypic interaction favorability. Lastly, interrogations of collective behavior via in vitro imaging of condensates indicated that MSL8^N, MSL9^N, and MSL10^N have sharply differing propensities for self‐assembly into condensates, both inherently and in response to salt, temperature, and molecular crowding. Taken together, these data establish the N‐termini of MSL channels as intrinsically disordered regions with distinct biophysical properties and the potential to respond uniquely to changes in their physiochemical environment.

White mold disease caused by a necrotrophic ascomycete pathogen Sclerotinia sclerotiorum results in serious economic losses of soybean yield in the USA. Lack of effective genetic resistance to this disease in soybean germplasm and increasing pathogen resistance to fungicides makes white mold difficult to manage. Small cysteine-rich antifungal peptides with multi-faceted modes of action hold potential for development as sustainable spray-on bio-fungicides. We have previously reported that GMA4CG_V6 peptide, a 17-amino acid variant of the MtDef4 defensin-derived peptide GMA4CG containing the active γ-core motif, exhibits potent antifungal activity against the gray mold fungal pathogen Botrytis cinerea in vitro and in planta . This peptide exhibited antifungal activity against an aggressive field isolate of S. sclerotiorum 555 in vitro. It markedly reduced white mold disease symptoms when applied to detached soybean leaves, pods, and stems. Spray-application on soybean plants provided robust control of the white mold disease. GMA4CG_V6 at sub-lethal concentrations reduced sclerotia production. It was also non-phytotoxic to soybean plants. Our results demonstrate that GMA4CG_V6 peptide has high potential for development as a bio-fungicide for white mold control in soybean.

Mechanosensitive (MS) ion channels are an evolutionarily conserved way for cells to sense mechanical forces and transduce them into ionic signals. The channel properties of Arabidopsis thaliana MscS-Like (MSL)10 have been well studied, but how MSL10 signals remains largely unknown. To uncover signaling partners of MSL10, we employed a proteomic screen and a forward genetic screen; both unexpectedly implicated endoplasmic reticulum–plasma membrane contact sites (EPCSs) in MSL10 function. The proteomic screen revealed that MSL10 associates with multiple proteins associated with EPCSs. Of these, only VAMP-associated proteins (VAP)27-1 and VAP27-3 interacted directly with MSL10. The forward genetic screen, for suppressors of a gain-of-function MSL10 allele (msl10-3G, MSL10^S640L), identified mutations in the synaptotagmin (SYT)5 and SYT7 genes. We also found that EPCSs were expanded in leaves of msl10-3G plants compared to the wild type. Taken together, these results indicate that MSL10 associates and functions with EPCS proteins, providing a new cell-level framework for understanding MSL10 signaling. In addition, placing a mechanosensory protein at EPCSs provides new insight into the function and regulation of this type of subcellular compartment.

Mechanosensitive (MS) ion channels are an evolutionarily conserved way for cells to sense mechanical forces and transduce them into ionic signals. The channel properties of Arabidopsis thaliana MscS-Like (MSL)10 have been well studied, but how MSL10 signals remains largely unknown. To uncover signaling partners of MSL10, we employed both a proteomic screen and a forward genetic screen; both unexpectedly implicated ER-plasma membrane contact sites (EPCSs) in MSL10 function. The proteomic screen revealed that MSL10 associates with multiple proteins associated with EPCSs. Of these, only VAMP-associated proteins (VAP)27-1 and VAP27-3 interacted directly with MSL10. The forward genetic screen, for suppressors of a gain-of-function MSL10 allele ( msl10-3G, MSL10 ^S640L ), identified mutations in the synaptotagmin (SYT)5 and SYT7 genes. We also found that EPCSs were expanded in leaves of msl10-3G plants compared to the wild type. Taken together, these results indicate that MSL10 can be found at EPCSs and functions there, providing a new cell-level framework for understanding MSL10 signaling. In addition, placing a mechanosensory protein at EPCS provides new insight into the function and regulation of this type of subcellular compartment.

The ability to sense and respond to physical forces is critical for the proper function of cells, tissues, and organisms across the evolutionary tree. Plants sense gravity, osmotic conditions, pathogen invasion, wind, and the presence of barriers in the soil, and dynamically integrate internal and external stimuli during every stage of growth and development. While the field of plant mechanobiology is growing, much is still poorly understood—including the interplay between mechanical and biochemical information at the single-cell level. In this review, we provide an overview of the mechanical properties of three main components of the plant cell and the mechanoperceptive pathways that link them, with an emphasis on areas of complexity and interaction. We discuss the concept of mechanical homeostasis, or “mechanostasis,” and examine the ways in which cellular structures and pathways serve to maintain it. We argue that viewing mechanics and mechanotransduction as emergent properties of the plant cell can be a useful conceptual framework for synthesizing current knowledge and driving future research.

Plants sense and respond to molecular signals associated with the presence of pathogens and their virulence factors. Mechanical signals generated during pathogenic invasion may also be important, but their contributions have rarely been studied. Here we investigate the potential role of a mechanosensitive ion channel, MscS-Like (MSL)10, in defense against the bacterial pathogen Pseudomonas syringae in Arabidopsis thaliana . We previously showed that overexpression of MSL10-GFP, phospho-mimetic versions of MSL10, and the gain-of-function allele msl10-3G all produce dwarfing, spontaneous cell death, and the hyperaccumulation of reactive oxygen species. These phenotypes are shared by many autoimmune mutants and are frequently suppressed by growth at high temperature in those lines. Here, we found that the same was true for all three MSL10 hypermorphs. In addition, we show that the SGT1/RAR1/HSP90 co-chaperone complex was required for dwarfing and ectopic cell death, PAD4 and SID2 were partially required, and the immune regulators EDS1 and NDR1 were dispensable. All MSL10 hypermorphs exhibited reduced susceptibility to infection by P. syringae strain Pto DC3000, Pto DC3000 expressing the avirulence genes avrRpt2 or avrRpm1 , but not Pto DC3000 hrpL , and showed an accelerated induction of PR1 expression compared to wild-type plants. Null msl10-1 mutants were delayed in PR1 induction and displayed modest susceptibility to infection by COR-deficient Pst . Finally, stomatal closure was reduced in msl10-1 loss-of-function mutants in response to Pst COR ⁻ . These data show that MSL10 modulates pathogen responses and begin to address the possibility that mechanical signals are exploited by the plant for pathogen perception.