The Cassava Source-Sink project: opportunities and challenges for crop improvement by metabolic engineering.

Cassava (Manihot esculenta Crantz) is one of the important staple foods in Sub-Saharan Africa. It produces starchy storage roots that provide food and income for several hundred million people, mainly in tropical agriculture zones. Increasing cassava storage root and starch yield is one of the major breeding targets with respect to securing the future food supply for the growing population of Sub-Saharan Africa. The Cassava Source-Sink (CASS) project aims to increase cassava storage root and starch yield by strategically integrating approaches from different disciplines. We present our perspective and progress on cassava as an applied research organism and provide insight into the CASS strategy, which can serve as a blueprint for the improvement of other root and tuber crops. Extensive profiling of different field-grown cassava genotypes generates information for leaf, phloem, and root metabolic and physiological processes that are relevant for biotechnological improvements. A multi-national pipeline for genetic engineering of cassava plants covers all steps from gene discovery, cloning, transformation, molecular and biochemical characterization, confined field trials, and phenotyping of the seasonal dynamics of shoot traits under field conditions. Together, the CASS project generates comprehensive data to facilitate conventional breeding strategies for high-yielding cassava genotypes. It also builds the foundation for genome-scale metabolic modelling aiming to predict targets and bottlenecks in metabolic pathways. This information is used to engineer cassava genotypes with improved source-sink relations and increased yield potential.

Alterations of plant architecture and phase transition by the phytoplasma virulence factor SAP11.

As key mediators linking developmental processes with plant immunity, TCP (TEOSINTE-BRANCHED, CYCLOIDEA, PROLIFERATION FACTOR 1 and 2) transcription factors have been increasingly shown to be targets of pathogenic effectors. We report here that TB/CYC (TEOSINTE-BRANCHED/CYCLOIDEA)-TCPs are destabilized by phytoplasma SAP11 effectors, leading to the proliferation of axillary meristems. Although a high degree of sequence diversity was observed among putative SAP11 effectors identified from evolutionarily distinct clusters of phytoplasmas, these effectors acquired fundamental activity in destabilizing TB/CYC-TCPs. In addition, we demonstrate that miR156/SPLs and miR172/AP2 modules, which represent key regulatory hubs involved in plant phase transition, were modulated by Aster Yellows phytoplasma strain Witches' Broom (AY-WB) protein SAP11. A late-flowering phenotype with significant changes in the expression of flowering-related genes was observed in transgenic Arabidopsis plants expressing SAP11AYWB. These morphological and molecular alterations were correlated with the ability of SAP11 effectors to destabilize CIN (CINCINNATA)-TCPs. Although not all putative SAP11 effectors display broad-spectrum activities in modulating morphological and physiological changes in host plants, they serve as core virulence factors responsible for the witches' broom symptom caused by phytoplasmas.

Phytoplasma SAP11 alters 3-isobutyl-2-methoxypyrazine biosynthesis in Nicotiana benthamiana by suppressing NbOMT1.

Phytoplasmas are bacterial phytopathogens that release virulence effectors into sieve cells and act systemically to affect the physiological and morphological state of host plants to promote successful pathogenesis. We show here that transgenic Nicotiana benthamiana lines expressing the secreted effector SAP11 from Candidatus Phytoplasma mali exhibit an altered aroma phenotype. This phenomenon is correlated with defects in the development of glandular trichomes and the biosynthesis of 3-isobutyl-2-methoxypyrazine (IBMP). IBMP is a volatile organic compound (VOC) synthesized by an O-methyltransferase, via a methylation step, from a non-volatile precursor, 3-isobutyl-2-hydroxypyrazine (IBHP). Based on comparative and functional genomics analyses, NbOMT1, which encodes an O-methyltransferase, was found to be highly suppressed in SAP11-transgenic plants. We further silenced NbOMT1 through virus-induced gene silencing and demonstrated that this enzyme influenced the accumulation of IBMP in N. benthamiana In vitro biochemical analyses also showed that NbOMT1 can catalyse IBHP O-methylation in the presence of S-adenosyl-L-methionine. Our study suggests that the phytoplasma effector SAP11 has the ability to modulate host VOC emissions. In addition, we also demonstrated that SAP11 destabilized TCP transcription factors and suppressed jasmonic acid responses in N. benthamiana These findings provide valuable insights into understanding how phytoplasma effectors influence plant volatiles.

Corrigendum: A promoter toolbox for tissue-specific expression supporting translational research in cassava (Manihot esculenta).

[This corrects the article DOI: 10.3389/fpls.2022.1042379.].

A promoter toolbox for tissue-specific expression supporting translational research in cassava (Manihot esculenta).

There is an urgent need to stimulate agricultural output in many tropical and subtropical countries of the world to combat hunger and malnutrition. The starchy crop cassava (Manihot esculenta), growing even under sub-optimal conditions, is a key staple food in these regions, providing millions of people with food. Cassava biotechnology is an important technique benefiting agricultural progress, but successful implementation of many biotechnological concepts depends on the availability of the right spatiotemporal expression tools. Yet, well-characterized cassava promoters are scarce in the public domain. In this study, we investigate the promoter activity and tissue specificity of 24 different promoter elements in stably transformed cassava plants. We show that many of the investigated promoters, especially from other species, have surprisingly low activity and/or tissue specificity, but feature several promoter sequences that can drive tissue-specific expression in either autotrophic-, transport- or storage tissues. We especially highlight pAtCAB1, pMePsbR, and pSlRBCS2 as strong and specific source promoters, pAtSUC2, pMeSWEET1-like, and pMeSUS1 as valuable tools for phloem and phloem parenchyma expression, and pStB33, pMeGPT, pStGBSS1, as well as pStPatatin Class I, as strong and specific promoters for heterotrophic storage tissues. We hope that the provided information and sequences prove valuable to the cassava community by contributing to the successful implementation of biotechnological concepts aimed at the improvement of cassava nutritional value and productivity.

Draft Genome Sequence of a 16SrII-A Subgroup Phytoplasma Associated with Purple Coneflower (Echinacea purpurea) Witches' Broom Disease in Taiwan.

The bacterial genus "Candidatus Phytoplasma" contains a group of insect-transmitted plant pathogens in the class Mollicutes. Here, we report a draft genome assembly and annotation of strain NCHU2014, which belongs to the 16SrII-A subgroup within this genus and is associated with purple coneflower witches' broom disease in Taiwan.

Arabidopsis HFR1 is a potential nuclear substrate regulated by the Xanthomonas type III effector XopD(Xcc8004).

XopDXcc8004, a type III effector of Xanthomonas campestris pv. campestris (Xcc) 8004, is considered a shorter version of the XopD, which lacks the N-terminal domain. To understand the functions of XopDXcc8004, in planta, a transgenic approach combined with inducible promoter to analyze the effects of XopDXcc8004 in Arabidopsis was done. Here, the expression of XopDXcc8004, in Arabidopsis elicited the accumulation of host defense-response genes. These molecular changes were dependent on salicylic acid and correlated with lesion-mimic phenotypes observed in XVE::XopDXcc8004 transgenic plants. Moreover, XopDXcc8004 was able to desumoylate HFR1, a basic helix-loop-helix transcription factor involved in photomorphogenesis, through SUMO protease activity. Interestingly, the hfr1-201 mutant increased the expression of host defense-response genes and displayed a resistance phenotype to Xcc8004. These data suggest that HFR1 is involved in plant innate immunity and is potentially regulated by XopDXcc8004.

Purification and biochemical characterization of Arabidopsis At-NEET, an ancient iron-sulfur protein, reveals a conserved cleavage motif for subcellular localization.

CDGSH iron-sulfur domain-containing proteins (CISDs) are newly discovered proteins with electron-accepting and electron-donating moieties. Although the CISDs of plants and animals show high sequence similarity in their CDGSH domain at the C-terminus, their N-terminal peptides have low sequence homology. Here, we show that At-NEET, a recently identified Arabidopsis CISD, contains a cleavable N-terminal peptide for chloroplast targeting, which is different from the uncleavable N-terminal peptide of mammal CISDs for mitochondrial outer membrane localization. Using affinity purification to isolate endogenous At-NEET, we identified a consensus sequence for the chloroplast transit peptide cleavage site of V-[R/K]↓A-E in At-NEET as well as other plant CISDs. Moreover, chloroplast subfractionation and immunogold labeling experiments showed that At-NEET localizes to the stroma of chloroplast. In addition, biochemical characterization revealed that At-NEET contains a conserved Cys(3)-His(1) ligand in the CDGSH domain, which is essential for coordination of 2Fe-2S clusters and protein folding. Our findings suggest that plant and animal CISDs contain an evolutionarily conserved CDGSH domain. However, they show different subcellular localization patterns that may result in distinct physiological functions.