high-throughput-image-based-phenotyping-for-determining-morphological

High Throughput Image-Based Phenotyping for Determining Morphological and Physiological Responses to Single and Combined Stresses in Potato

High throughput image-based phenotyping is a powerful tool to non-invasively determine the development and performance of plants under specific conditions ...

Friedrich-Alexander Universität Erlangen-Nürnberg

Cell wall-bound invertase (cw-Inv) plays an important role in carbohydrate partitioning and regulation of sink-source interaction. There is increasing evidence that pathogens interfere with sink-source interaction, and induction of cw-Inv activity has frequently been shown in response to pathogen infection. To investigate the role of cw-Inv, transgenic tomato (Solanum lycopersicum) plants silenced for the major leaf cw-Inv isoforms were generated and analyzed during normal growth and during the compatible interaction with Xanthomonas campestris pv vesicatoria. Under normal growth conditions, activities of sucrolytic enzymes as well as photosynthesis and respiration were unaltered in the transgenic plants compared with wild-type plants. However, starch levels of source leaves were strongly reduced, which was most likely caused by an enhanced sucrose exudation rate. Following X. campestris pv vesicatoria infection, cw-Inv-silenced plants showed an increased sucrose to hexose ratio in the apoplast of leaves. Symptom development, inhibition of photosynthesis, and expression of photosynthetic genes were clearly delayed in transgenic plants compared with wild-type plants. In addition, induction of senescence-associated and pathogenesis-related genes observed in infected wild-type plants was abolished in cw-Inv-silenced tomato lines. These changes were not associated with decreased bacterial growth. In conclusion, cw-Inv restricts carbon export from source leaves and regulates the sucrose to hexose ratio in the apoplast. Furthermore, an increased apoplastic hexose to sucrose ratio can be linked to inhibition of photosynthesis and induction of pathogenesis-related gene expression but does not significantly influence bacterial growth. Indirectly, bacteria may benefit from low invertase activity, since the longevity of host cells is raised and basal defense might be dampened.

Cell wall-bound invertase limits sucrose export and is involved in symptom development and inhibition of photosynthesis during compatible interaction between tomato and Xanthomonas campestris pv vesicatoria.

The phytopathogenic bacterium Xanthomonas campestris pv. vesicatoria uses the type III secretion system (T3SS) to inject effector proteins into cells of its Solanaceous host plants. It is generally assumed that these effectors manipulate host pathways to favor bacterial replication and survival. However, the molecular mechanisms by which type III effectors suppress host defense responses are far from being understood. Based on sequence similarity, Xanthomonas outer protein J (XopJ) is a member of the YopJ/AvrRxv family of SUMO peptidases and acetyltranferases, although its biochemical activity has not yet been demonstrated. Confocal laser scanning microscopy revealed that green fluorescent protein (GFP) fusions of XopJ are targeted to the plasma membrane when expressed in plant cells, which most likely involves N-myristoylation. In contrast to a XopJ(C235A) mutant disrupted in the catalytic triad sequence, the wild-type effector GFP fusion protein was also localized in vesicle-like structures colocalizing together with a Golgi marker protein, suggesting an effect of XopJ on vesicle trafficking. To explore an effect of XopJ on protein secretion, we used a GFP-based secretion assay. When a secreted (sec)GFP marker was coexpressed with XopJ in leaves of Nicotiana benthamiana, GFP fluorescence was retained in reticulate structures. In contrast, in plant cells expressing secGFP alone or along with the XopJ(C235A) mutant, no GFP fluorescence accumulated within the cells. Moreover, coexpressing secGFP together with XopJ led to a reduced accumulation of secGFP within the apoplastic fluid of N. benthamiana leaves, further showing that XopJ affects protein secretion. Transgenic expression of XopJ in Arabidopsis suppressed callose deposition elicited by a T3SS-negative mutant of Pseudomonas syringae pv. tomato DC3000. A role of XopJ in the inhibition of cell wall-based defense responses is discussed.

The Xanthomonas campestris pv. vesicatoria type III effector protein XopJ inhibits protein secretion: evidence for interference with cell wall-associated defense responses.

Reactivation of dormant meristems is of central importance for plant fitness and survival. Due to their large meristem size, potato (Solanum tuberosum) tubers serve as a model system to study the underlying molecular processes. The phytohormones cytokinins (CK) and gibberellins (GA) play important roles in releasing potato tuber dormancy and promoting sprouting, but their mode of action in these processes is still obscure. Here, we established an in vitro assay using excised tuber buds to study the dormancy-releasing capacity of GA and CK and show that application of gibberellic acid (GA(3)) is sufficient to induce sprouting. In contrast, treatment with 6-benzylaminopurine induced bud break but did not support further sprout growth unless GA(3) was administered additionally. Transgenic potato plants expressing Arabidopsis (Arabidopsis thaliana) GA 20-oxidase or GA 2-oxidase to modify endogenous GA levels showed the expected phenotypical changes as well as slight effects on tuber sprouting. The isopentenyltransferase (IPT) from Agrobacterium tumefaciens and the Arabidopsis cytokinin oxidase/dehydrogenase1 (CKX) were exploited to modify the amounts of CK in transgenic potato plants. IPT expression promoted earlier sprouting in vitro. Strikingly, CKX-expressing tubers exhibited a prolonged dormancy period and did not respond to GA(3). This supports an essential role of CK in terminating tuber dormancy and indicates that GA is not sufficient to break dormancy in the absence of CK. GA(3)-treated wild-type and CKX-expressing tuber buds were subjected to a transcriptome analysis that revealed transcriptional changes in several functional groups, including cell wall metabolism, cell cycle, and auxin and ethylene signaling, denoting events associated with the reactivation of dormant meristems.

Reactivation of meristem activity and sprout growth in potato tubers require both cytokinin and gibberellin.

Xanthomonas campestris pv. vesicatoria (Xcv) possess a type 3 secretion system (T3SS) to deliver effector proteins into its Solanaceous host plants. These proteins are involved in suppression of plant defense and in reprogramming of plant metabolism to favour bacterial propagation. There is increasing evidence that hexoses contribute to defense responses. They act as substrates for metabolic processes and as metabolic semaphores to regulate gene expression. Especially an increase in the apoplastic hexose-to-sucrose ratio has been suggested to strengthen plant defense. This shift is brought about by the activity of cell wall-bound invertase (cw-Inv). We examined the possibility that Xcv may employ type 3 effector (T3E) proteins to suppress cw-Inv activity during infection. Indeed, pepper leaves infected with a T3SS-deficient Xcv strain showed a higher level of cw-Inv mRNA and enzyme activity relative to Xcv wild type infected leaves. Higher cw-Inv activity was paralleled by an increase in hexoses and mRNA abundance for the pathogenesis-related gene PRQ. These results suggest that Xcv suppresses cw-Inv activity in a T3SS-dependent manner, most likely to prevent sugar-mediated defense signals. To identify Xcv T3Es that regulate cw-Inv activity, a screen was performed with eighteen Xcv strains, each deficient in an individual T3E. Seven Xcv T3E deletion strains caused a significant change in cw-Inv activity compared to Xcv wild type. Among them, Xcv lacking the xopB gene (Xcv ΔxopB) caused the most prominent increase in cw-Inv activity. Deletion of xopB increased the mRNA abundance of PRQ in Xcv ΔxopB-infected pepper leaves, but not of Pti5 and Acre31, two PAMP-triggered immunity markers. Inducible expression of XopB in transgenic tobacco inhibited Xcv-mediated induction of cw-Inv activity observed in wild type plants and resulted in severe developmental phenotypes. Together, these data suggest that XopB interferes with cw-Inv activity in planta to suppress sugar-enhanced defense responses during Xcv infection.

Regulation of cell wall-bound invertase in pepper leaves by Xanthomonas campestris pv. vesicatoria type three effectors.

The interaction between Clavibacter michiganensis subsp. michiganensis with its host, the tomato plant (Solanum lycopersicum), is poorly understood and only few virulence factors are known. While studying of the bacteria in planta is time-consuming and difficult, the analysis in vitro would facilitate research. Therefore, a xylem mimicking medium (XMM) for C. michiganensis subsp. michiganensis was established in this study based on an apoplast medium for Xanthomonas campestris pv. vesicatoria. In contrast to the apoplast medium, XMM contains no sugars, but amino acids which serve as nitrogen and carbon source. As a result, growth in XMM induced transcriptional changes of genes encoding putative sugar, amino acid and iron uptake systems. In summary, mRNA levels of about 8% of all C. michiganensis subsp. michiganensis genes were changed when XMM-grown bacteria were compared to M9 minimal medium-grown cells. Almost no transcriptional changes of genes encoding hydrolytic enzymes were detected, leading to the idea that XMM reflects the situation in the beginning of infection and therefore allows the characterization of virulence factors in this early stage of infection. The addition of the plant wound substance acetosyringone to the XMM medium led to a change in transcript amount, including genes coding for proteins involved in protein transport, iron uptake and regulation processes.

Genome-wide transcriptome analysis of Clavibacter michiganensis subsp. michiganensis grown in xylem mimicking medium.

Following tuber induction, potato tubers undergo a period of dormancy during which visible bud growth is inhibited. The length of the dormancy period is under environmental, physiological and hormonal control. Sucrose availability is one prerequisite for bud break. In the absence of sucrose, no bud break occurs. Thus, sucrose is likely to serve as nutrient and signal molecule at the same time. The mode of sucrose sensing is only vaguely understood, but most likely involves trehalose-6-phosphate and SnRK1 signalling networks. This conclusion is supported by the observation that ectopically manipulation of trehalose-6-phosphate levels influences the length of the dormancy period. Once physiological competence is achieved, sprouting is controlled by the level of phytohormones. Two phytohormones, ABA and ethylene, are supposed to suppress tuber sprouting; however, the exact role of ethylene remains to be elucidated. Cytokinins and gibberellins are required for bud break and sprout growth, respectively. The fifth classical phytohormone, auxin, seems to play a role in vascular development. During the dormancy period, buds are symplastically isolated, which changes during bud break. In parallel to the establishment of symplastic connectivity, vascular tissue develops below the growing bud most likely to support the outgrowing sprout with assimilates mobilised in parenchyma cells. Sprouting leads to major quality losses of stored potato tubers. Therefore, control of tuber sprouting is a major objective in potato breeding. Although comparative transcriptome analysis revealed a large number of genes differentially expressed in growing versus dormant buds, no master-regulator of potato tuber sprouting has been identified so far.

Regulation of potato tuber sprouting.

As other bacteria, Mycobacterium smegmatis needs adaption mechanisms to cope with changing nitrogen sources and to survive situations of nitrogen starvation. In the study presented here, transcriptome analyses were used to characterize the response of the bacterium to nitrogen starvation and to elucidate the role of specific transcriptional regulators.

Nitrogen starvation-induced transcriptome alterations and influence of transcription regulator mutants in Mycobacterium smegmatis.

Starch is the principle constituent of potato tubers and is of considerable importance for food and non-food applications. Its metabolism has been subject of extensive research over the past decades. Despite its importance, a description of the complete inventory of genes involved in starch metabolism and their genome organization in potato plants is still missing. Moreover, mechanisms regulating the expression of starch genes in leaves and tubers remain elusive with regard to differences between transitory and storage starch metabolism, respectively. This study aimed at identifying and mapping the complete set of potato starch genes, and to study their expression pattern in leaves and tubers using different sets of transcriptome data. Moreover, we wanted to uncover transcription factors co-regulated with starch accumulation in tubers in order to get insight into the regulation of starch metabolism.

Genome-wide analysis of starch metabolism genes in potato (Solanum tuberosum L.).

Plants have evolved a multitude of strategies to adjust their growth according to external and internal signals. Interconnected metabolic and phytohormonal signalling networks allow adaption to changing environmental and developmental conditions and ensure the survival of species in fluctuating environments. In agricultural ecosystems, many of these adaptive responses are not required or may even limit crop yield, as they prevent plants from realizing their fullest potential. By lifting source and sink activities to their maximum, massive yield increases can be foreseen, potentially closing the future yield gap resulting from an increasing world population and the transition to a carbon-neutral economy. To do so, a better understanding of the interplay between metabolic and developmental processes is required. In the past, these processes have been tackled independently from each other, but coordinated efforts are required to understand the fine mechanics of source-sink relations and thus optimize crop yield. Here, we describe approaches to design high-yielding crop plants utilizing strategies derived from current metabolic concepts and our understanding of the molecular processes determining sink development.

Synchronization of developmental, molecular and metabolic aspects of source-sink interactions.

The obligate intracellular pathogen Coxiella burnetii is the causative agent of the zoonosis Q fever. C. burnetii infection can have severe outcomes due to the development of chronic infection. To establish and maintain an infection, C. burnetii depends on a functional type IVB secretion system (T4BSS) and, thus, on the translocation of effector proteins into the host cell. Here, we showed that the C. burnetii T4BSS effector protein CaeB targets the conserved endoplasmatic reticulum (ER) stress sensor IRE1 during ER stress in mammalian and plant cells. CaeB-induced upregulation of IRE1 RNase activity was essential for CaeB-mediated inhibition of ER stress-induced cell death. Our data reveal a novel role for CaeB in ER stress signalling modulation and demonstrate that CaeB is involved in pathogenicity in vivo. Furthermore, we provide evidence that C. burnetii infection leads to modulation of the ER stress sensors IRE1 and PERK, but not ATF6 during ER stress. While the upregulation of the RNase activity of IRE1 during ER stress depends on CaeB, modulation of PERK is CaeB independent, suggesting that C. burnetii encodes several factors influencing ER stress during infection.

The Coxiella burnetii effector protein CaeB modulates endoplasmatic reticulum (ER) stress signalling and is required for efficient replication in Galleria mellonella.

Root and tuber crops have been an important part of human nutrition since the early days of humanity, providing us with essential carbohydrates, proteins, and vitamins. Today, they are especially important in tropical and subtropical regions of the world, where they help to feed an ever-growing population. Early induction and storage organ size are important agricultural traits, as they determine yield over time. During potato tuberization, environmental and metabolic status are sensed, ensuring proper timing of tuberization mediated by phloem-mobile signals. Coordinated cellular restructuring and expansion growth, as well as controlled storage metabolism in the tuber, are executed. This review summarizes our current understanding of potato tuber development and highlights similarities and differences to important tuberous root crop species like sweetpotato and cassava. Finally, we point out knowledge gaps that need to be filled before a complete picture of storage organ development can emerge.

Tuber and Tuberous Root Development.

Inflammatory bowel diseases (IBD) are characterized by mucosal inflammation and sequential fibrosis formation, but the exact role of the hyperactive NLRP3 inflammasome in these processes is unclear. Thus, we studied the expression and function of the NLRP3 inflammasome in the context of inflammation and fibrosis in IBD.

NLRP3 Inhibition Leads to Impaired Mucosal Fibroblast Function in Patients with Inflammatory Bowel Diseases.

Potatoes (Solanum tuberosum L.) are a fundamental staple for millions of people worldwide. They provide essential amino acids, vitamins, and starch - a vital component of the human diet, providing energy and serving as a source of fiber. Unfortunately, global warming is posing a severe threat to this crop, leading to significant yield losses, and thereby endangering global food security. Industrial agriculture traditionally relies on excessive nitrogen (N) fertilization to boost yields. However, it remains uncertain whether this is effective in combating heat-related yield losses of potato. Therefore, our study aimed to investigate the combinatory effects of heat stress and N fertilization on potato tuber formation. We demonstrate that N levels and heat significantly impact tuber development. The combination of high N and heat delays tuberization, while N deficiency initiates early tuberization, likely through starvation-induced signals, independent of SELF-PRUNING 6A (SP6A), a critical regulator of tuberization. We also found that high N levels in combination with heat reduce tuber yield rather than improve it. However, our study revealed that SP6A overexpression can promote tuberization under these inhibiting conditions. By utilizing the excess of N for accumulating tuber biomass, SP6A overexpressing plants exhibit a shift in biomass distribution towards the tubers. This results in an increased yield compared to wild-type plants. Our results highlight the role of SP6A overexpression as a viable strategy for ensuring stable potato yields in the face of global warming. As such, our findings provide insights into the complex factors impacting potato crop productivity.

Article	Year
Cell wall-bound invertase limits sucrose export and is involved in symptom development and inhibition of photosynthesis during compatible interaction between tomato and Xanthomonas campestri... Plant physiology\| PubMed ID: 18784281	2008
The Xanthomonas campestris pv. vesicatoria type III effector protein XopJ inhibits protein secretion: evidence for interference with cell wall-associated defense responses. Molecular plant-microbe interactions : MPMI\| PubMed ID: 19445590	2009
Reactivation of meristem activity and sprout growth in potato tubers require both cytokinin and gibberellin. Plant physiology\| PubMed ID: 21163959	2011
Regulation of cell wall-bound invertase in pepper leaves by Xanthomonas campestris pv. vesicatoria type three effectors. PloS one\| PubMed ID: 23272161	2012
Genome-wide transcriptome analysis of Clavibacter michiganensis subsp. michiganensis grown in xylem mimicking medium. Journal of biotechnology\| PubMed ID: 24060828	2013
Regulation of potato tuber sprouting. Planta\| PubMed ID: 24100410	2014
Nitrogen starvation-induced transcriptome alterations and influence of transcription regulator mutants in Mycobacterium smegmatis. BMC research notes\| PubMed ID: 24266988	2013
Genome-wide analysis of starch metabolism genes in potato (Solanum tuberosum L.). BMC genomics\| PubMed ID: 28056783	2017
Synchronization of developmental, molecular and metabolic aspects of source-sink interactions. Nature plants\| PubMed ID: 32042154	2020
The Coxiella burnetii effector protein CaeB modulates endoplasmatic reticulum (ER) stress signalling and is required for efficient replication in Galleria mellonella. Cellular microbiology\| PubMed ID: 33355405	2021
Tuber and Tuberous Root Development. Annual review of plant biology\| PubMed ID: 33788583	2021
NLRP3 Inhibition Leads to Impaired Mucosal Fibroblast Function in Patients with Inflammatory Bowel Diseases. Journal of Crohn's & colitis\| PubMed ID: 37748021	2024
Yield reduction caused by elevated temperatures and high nitrogen fertilization is mitigated by SP6A overexpression in potato (Solanum tuberosum L.). The Plant journal : for cell and molecular biology\| PubMed ID: 38334712	2024

Sophia Sonnewald

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