First report of Geotrichum candidum causing rubbery rot of potato (Solanum tuberosum) in Michigan.

In April 2018, rotted 'Lamoka' tubers were received from a commercial storage facility (<1% incidence) in St. Joseph County, Michigan by the MSU Potato & Sugar Beet Pathology and Plant & Pest Diagnostics programs. Dense circular colonies of white fungal-like growth were observed on the surface of the tubers, and internal tissues were watery and spongy with gray to brown discoloration (Supplemental Figure 1). Tubers had a strong, sweet alcoholic odor. External and internal tuber tissues were surface disinfested in 0.825% sodium hypochlorite for 1 min, rinsed twice in sterile distilled water, blotted on sterile filter paper, and placed onto 1.5% (w/v) water agar (WA). After 3 days at 21-24°C and ambient light conditions, septate, branched mycelia and hyaline, cylindrical, single-celled conidia 5.2-8.9 µm x 3.6-5.2 µm (n=20 arthrospores) were observed singly or in chains (Supplemental Figure 2A&B). On potato dextrose agar (PDA), colonies were white, circular, and dense (Supplemental Figure 2C). These observations matched morphological descriptions of Geotrichum candidum (Carmichael 1957). No Pythium or Phytophthora spp. were detected. A mono-conidial isolate of the fungus was obtained and maintained on PDA. DNA was extracted from mycelia using a DNeasy plant mini kit (QIAGEN). Fragments of the internal transcribed spacer (ITS) and 18S ribosomal RNA gene regions were amplified using primers ITS1F/4 primers and NS3/8, respectively (White et al. 1990). Purified PCR products (QIAquick PCR purification kit, QIAGEN) were submitted for Sanger sequencing at the Genomics Research Technology Support Facility (East Lansing, MI). The ITS1F/4 and NS3/8 consensus sequences (OP142324 and OP153873) aligned with GenBank accessions of G. candidum KY103456.1 (100% identity) and JF262193.1 (99.75% identity), respectively. Healthy 'Lamoka' tubers were rinsed with tap water, surface disinfested in 0.825% sodium hypochlorite for 15 min, rinsed twice in sterile distilled water, and blotted dry on sterile paper towel. Ten tubers were inoculated by placing 10-mm diameter fully colonized agar plugs, excised from the margin of a 9-day-old PDA culture, onto the surface of each tuber (Duellman et al. 2021). Ten tubers were mock-inoculated using sterile PDA. Tubers were placed in a moist chamber and incubated in the dark at room temperature. After nine days, inoculated tubers exhibited white colony growth on tuber surfaces and an alcoholic scent was present. After 27 days, internal tissues were rubbery, but no discoloration was observed. No rubbery rot symptoms were observed on the control tubers. Samples were excised 1 cm laterally from and vertically beneath the inoculation site. Tissues were surface disinfested as described above and plated on 1.5% WA. After 9 days, a Geotrichum sp. identical to the original isolate was confirmed in 50% of samples from inoculated tubers. No Geotrichum sp. were detected from mock-inoculated tubers. Since 2018, G. candidum has been confirmed in commercial storages in three counties in the Lower Peninsula (incidences up to 1-2%). Geotrichum candidum was recently reported causing rubbery rot of potato in Idaho (Duellman et al. 2021); however, to our knowledge this is the first report of rubbery rot in Michigan. Despite increasing detection frequencies, incidences remain low and spread in storage appears limited. Seed decay leading to stand loss (incidence 1-3%) was observed after planting infected lots, which should be avoided or minimized.

Limitations to photosynthesis by proton motive force-induced photosystem II photodamage.

The thylakoid proton motive force (pmf) generated during photosynthesis is the essential driving force for ATP production; it is also a central regulator of light capture and electron transfer. We investigated the effects of elevated pmf on photosynthesis in a library of Arabidopsis thaliana mutants with altered rates of thylakoid lumen proton efflux, leading to a range of steady-state pmf extents. We observed the expected pmf-dependent alterations in photosynthetic regulation, but also strong effects on the rate of photosystem II (PSII) photodamage. Detailed analyses indicate this effect is related to an elevated electric field (Δψ) component of the pmf, rather than lumen acidification, which in vivo increased PSII charge recombination rates, producing singlet oxygen and subsequent photodamage. The effects are seen even in wild type plants, especially under fluctuating illumination, suggesting that Δψ-induced photodamage represents a previously unrecognized limiting factor for plant productivity under dynamic environmental conditions seen in the field.

Dynamic Environmental Photosynthetic Imaging Reveals Emergent Phenotypes.

Understanding and improving the productivity and robustness of plant photosynthesis requires high-throughput phenotyping under environmental conditions that are relevant to the field. Here we demonstrate the dynamic environmental photosynthesis imager (DEPI), an experimental platform for integrated, continuous, and high-throughput measurements of photosynthetic parameters during plant growth under reproducible yet dynamic environmental conditions. Using parallel imagers obviates the need to move plants or sensors, reducing artifacts and allowing simultaneous measurement on large numbers of plants. As a result, DEPI can reveal phenotypes that are not evident under standard laboratory conditions but emerge under progressively more dynamic illumination. We show examples in mutants of Arabidopsis of such "emergent phenotypes" that are highly transient and heterogeneous, appearing in different leaves under different conditions and depending in complex ways on both environmental conditions and plant developmental age. These emergent phenotypes appear to be caused by a range of phenomena, suggesting that such previously unseen processes are critical for plant responses to dynamic environments.

CisSERS: Customizable In Silico Sequence Evaluation for Restriction Sites.

High-throughput sequencing continues to produce an immense volume of information that is processed and assembled into mature sequence data. Data analysis tools are urgently needed that leverage the embedded DNA sequence polymorphisms and consequent changes to restriction sites or sequence motifs in a high-throughput manner to enable biological experimentation. CisSERS was developed as a standalone open source tool to analyze sequence datasets and provide biologists with individual or comparative genome organization information in terms of presence and frequency of patterns or motifs such as restriction enzymes. Predicted agarose gel visualization of the custom analyses results was also integrated to enhance the usefulness of the software. CisSERS offers several novel functionalities, such as handling of large and multiple datasets in parallel, multiple restriction enzyme site detection and custom motif detection features, which are seamlessly integrated with real time agarose gel visualization. Using a simple fasta-formatted file as input, CisSERS utilizes the REBASE enzyme database. Results from CisSERS enable the user to make decisions for designing genotyping by sequencing experiments, reduced representation sequencing, 3'UTR sequencing, and cleaved amplified polymorphic sequence (CAPS) molecular markers for large sample sets. CisSERS is a java based graphical user interface built around a perl backbone. Several of the applications of CisSERS including CAPS molecular marker development were successfully validated using wet-lab experimentation. Here, we present the tool CisSERS and results from in-silico and corresponding wet-lab analyses demonstrating that CisSERS is a technology platform solution that facilitates efficient data utilization in genomics and genetics studies.

Defects in the Expression of Chloroplast Proteins Leads to H2O2 Accumulation and Activation of Cyclic Electron Flow around Photosystem I.

We describe a new member of the class of mutants in Arabidopsis exhibiting high rates of cyclic electron flow around photosystem I (CEF), a light-driven process that produces ATP but not NADPH. High cyclic electron flow 2 (hcef2) shows strongly increased CEF activity through the NADPH dehydrogenase complex (NDH), accompanied by increases in thylakoid proton motive force (pmf), activation of the photoprotective qE response, and the accumulation of H2O2. Surprisingly, hcef2 was mapped to a non-sense mutation in the TADA1 (tRNA adenosine deaminase arginine) locus, coding for a plastid targeted tRNA editing enzyme required for efficient codon recognition. Comparison of protein content from representative thylakoid complexes, the cytochrome bf complex, and the ATP synthase, suggests that inefficient translation of hcef2 leads to compromised complex assembly or stability leading to alterations in stoichiometries of major thylakoid complexes as well as their constituent subunits. Altered subunit stoichiometries for photosystem I, ratios and properties of cytochrome bf hemes, and the decay kinetics of the flash-induced thylakoid electric field suggest that these defect lead to accumulation of H2O2 in hcef2, which we have previously shown leads to activation of NDH-related CEF. We observed similar increases in CEF, as well as increases in H2O2 accumulation, in other translation defective mutants. This suggests that loss of coordination in plastid protein levels lead to imbalances in photosynthetic energy balance that leads to an increase in CEF. These results taken together with a large body of previous observations, support a general model in which processes that lead to imbalances in chloroplast energetics result in the production of H2O2, which in turn activates CEF. This activation could be from either H2O2 acting as a redox signal, or by a secondary effect from H2O2 inducing a deficit in ATP.

Activation of cyclic electron flow by hydrogen peroxide in vivo.

Cyclic electron flow (CEF) around photosystem I is thought to balance the ATP/NADPH energy budget of photosynthesis, requiring that its rate be finely regulated. The mechanisms of this regulation are not well understood. We observed that mutants that exhibited constitutively high rates of CEF also showed elevated production of H2O2. We thus tested the hypothesis that CEF can be activated by H2O2 in vivo. CEF was strongly increased by H2O2 both by infiltration or in situ production by chloroplast-localized glycolate oxidase, implying that H2O2 can activate CEF either directly by redox modulation of key enzymes, or indirectly by affecting other photosynthetic processes. CEF appeared with a half time of about 20 min after exposure to H2O2, suggesting activation of previously expressed CEF-related machinery. H2O2-dependent CEF was not sensitive to antimycin A or loss of PGR5, indicating that increased CEF probably does not involve the PGR5-PGRL1 associated pathway. In contrast, the rise in CEF was not observed in a mutant deficient in the chloroplast NADPH:PQ reductase (NDH), supporting the involvement of this complex in CEF activated by H2O2. We propose that H2O2 is a missing link between environmental stress, metabolism, and redox regulation of CEF in higher plants.

Plastidial transporters KEA1, -2, and -3 are essential for chloroplast osmoregulation, integrity, and pH regulation in Arabidopsis.

Multiple K(+) transporters and channels and the corresponding mutants have been described and studied in the plasma membrane and organelle membranes of plant cells. However, knowledge about the molecular identity of chloroplast K(+) transporters is limited. Potassium transport and a well-balanced K(+) homeostasis were suggested to play important roles in chloroplast function. Because no loss-of-function mutants have been identified, the importance of K(+) transporters for chloroplast function and photosynthesis remains to be determined. Here, we report single and higher-order loss-of-function mutants in members of the cation/proton antiporters-2 antiporter superfamily KEA1, KEA2, and KEA3. KEA1 and KEA2 proteins are targeted to the inner envelope membrane of chloroplasts, whereas KEA3 is targeted to the thylakoid membrane. Higher-order but not single mutants showed increasingly impaired photosynthesis along with pale green leaves and severely stunted growth. The pH component of the proton motive force across the thylakoid membrane was significantly decreased in the kea1kea2 mutants, but increased in the kea3 mutant, indicating an altered chloroplast pH homeostasis. Electron microscopy of kea1kea2 leaf cells revealed dramatically swollen chloroplasts with disrupted envelope membranes and reduced thylakoid membrane density. Unexpectedly, exogenous NaCl application reversed the observed phenotypes. Furthermore, the kea1kea2 background enables genetic analyses of the functional significance of other chloroplast transporters as exemplified here in kea1kea2Na(+)/H(+) antiporter1 (nhd1) triple mutants. Taken together, the presented data demonstrate a fundamental role of inner envelope KEA1 and KEA2 and thylakoid KEA3 transporters in chloroplast osmoregulation, integrity, and ion and pH homeostasis.

A guanine nucleotide exchange factor for Rab5 proteins is essential for intracellular transport of the proglutelin from the Golgi apparatus to the protein storage vacuole in rice endosperm.

Rice (Oryza sativa) glutelins are synthesized on the endoplasmic reticulum as a precursor, which are then transported via the Golgi to protein storage vacuoles (PSVs), where they are proteolytically processed into acidic and basic subunits. The glutelin precursor mutant6 (glup6) accumulates abnormally large amounts of proglutelin. Map-base cloning studies showed that glup6 was a loss-of-function mutant of guanine nucleotide exchange factor (GEF), which activates Rab GTPase, a key regulator of membrane trafficking. Immunofluorescence studies showed that the transport of proglutelins and α-globulins to PSV was disrupted in glup6 endosperm. Secreted granules of glutelin and α-globulin were readily observed in young glup6 endosperm, followed by the formation of large dilated paramural bodies (PMBs) containing both proteins as the endosperm matures. The PMBs also contained membrane biomarkers for the Golgi and prevacuolar compartment as well as the cell wall component, ß-glucan. Direct evidence was gathered showing that GLUP6/GEF activated in vitro GLUP4/Rab5 as well as several Arabidopsis (Arabidopsis thaliana) Rab5 isoforms to the GTP-bound form. Therefore, loss-of-function mutations in GEF or Rab5 disrupt the normal transport of proglutelin from the Golgi to PSVs, resulting in the initial extracellular secretion of these proteins followed, in turn, by the formation of PMBs. Overall, our results indicate that GLUP6/GEF is the activator of Rab5 GTPase and that the cycling of GTP- and GDP-bound forms of this regulatory protein is essential for the intracellular transport of proglutelin and α-globulin from the Golgi to PSVs and in the maintenance of the general structural organization of the endomembrane system in rice seeds.

RNA targeting to a specific ER sub-domain is required for efficient transport and packaging of α-globulins to the protein storage vacuole in developing rice endosperm.

Studies focusing on the targeting of RNAs that encode rice storage proteins, prolamines and glutelins to specific sub-domains of the endoplasmic reticulum (ER), as well as mis-localization studies of other storage protein RNAs, indicate a close relationship between the ER site of RNA translation and the final site of protein deposition in the endomembrane system in developing rice endosperm. In addition to prolamine and glutelin, rice accumulates smaller amounts of α-globulins, which are deposited together with glutelin in the protein storage vacuole (PSV). In situ RT-PCR analysis revealed that α-globulin RNAs are not distributed to the cisternal ER as expected for a PSV-localized protein, but instead are targeted to the protein body-ER (PB-ER) by a regulated process requiring cis-sorting sequences. Sequence alignments with putative maize δ-zein cis-localization elements identified several candidate regulatory sequences that may be responsible for PB-ER targeting. Immunocytochemical analysis confirmed the presence of α-globulin on the periphery of the prolamine protein bodies and packaging in Golgi-associated dense vesicles, as well as deposition and storage within peripheral regions of the PSV. Mis-targeting of α-globulin RNAs to the cisternal ER dramatically alters the spatial arrangement of α-globulin and glutelin within the PSV, with the accompanying presence of numerous small α-globulin particles in the cytoplasm. These results indicate that α-globulin RNA targeting to the PB-ER sub-domain is essential for efficient transport of α-globulins to the PSV and its spatial arrangement in the PSV. Such RNA localization prevents potential deleterious protein-protein interactions, in addition to performing a role in protein targeting.

The small GTPase Rab5a is essential for intracellular transport of proglutelin from the Golgi apparatus to the protein storage vacuole and endosomal membrane organization in developing rice endosperm.

Rice (Oryza sativa) glutelins are synthesized on the endoplasmic reticulum as larger precursors, which are then transported via the Golgi to the protein storage vacuole (PSV), where they are processed into acidic and basic subunits. Three independent glutelin precursor mutant4 (glup4) rice lines, which accumulated elevated levels of proglutelin over the wild type, were identified as loss-of-function mutants of Rab5a, the small GTPase involved in vesicular membrane transport. In addition to the plasma membrane, Rab5a colocalizes with glutelins on the Golgi apparatus, Golgi-derived dense vesicles, and the PSV, suggesting that Rab5a participates in the transport of the proglutelin from the Golgi to the PSV. This spatial distribution pattern was dramatically altered in the glup4 mutants. Numerous smaller protein bodies containing glutelin and α-globulin were evident, and the proteins were secreted extracellularly. Moreover, all three independent glup4 allelic lines displayed the novel appearance of a large dilated, structurally complex paramural body containing proglutelins, α-globulins, membrane biomarkers for the Golgi apparatus, prevacuolar compartment, PSV, and the endoplasmic reticulum luminal chaperones BiP and protein disulfide isomerase as well as ß-glucan. These results indicate that the formation of the paramural bodies in glup4 endosperm was due to a significant disruption of endocytosis and membrane vesicular transport by Rab5a loss of function. Overall, Rab5a is required not only for the intracellular transport of proglutelins from the Golgi to the PSV in rice endosperm but also in the maintenance of the general structural organization of the endomembrane system in developing rice seeds.

Protein disulfide isomerase like 1-1 participates in the maturation of proglutelin within the endoplasmic reticulum in rice endosperm.

The rice esp2 mutation was previously characterized by the abnormal accumulation of elevated levels of proglutelin and the absence of an endosperm-specific protein disulfide isomerase like (PDIL1-1). Here we show that Esp2 is the structural gene for PDIL1-1 and that this lumenal chaperone is asymmetrically distributed within the cortical endoplasmic reticulum (ER) and largely restricted to the cisternal ER. Temporal studies indicate that PDIL1-1 is essential for the maturation of proglutelin only when its rate of synthesis significantly exceeds its export from the ER, a condition resulting in its build up in the ER lumen and the induction of ER quality control processes which lower glutelin levels as well as those of the other storage proteins. As proglutelin is initially synthesized on the cisternal ER, its deposition within prolamine protein bodies in esp2 suggests that PDIL1-1 helps retain proglutelin in the cisternal ER lumen until it attains competence for ER export and, thereby, indirectly preventing heterotypic interactions with prolamine polypeptides.