The Xanthomonas citri effector protein PthA interacts with citrus proteins involved in nuclear transport, protein folding and ubiquitination associated with DNA repair

P>Xanthomonas axonopodis pv. citri utilizes the type III effector protein PthA to modulate host transcription to promote citrus canker. PthA proteins belong to the AvrBs3/PthA family and carry a domain comprising tandem repeats of 34 amino acids that mediates protein-protein and protein-DNA interactions. We show here that variants of PthAs from a single bacterial strain localize to the nucleus of plant cells and form homo- and heterodimers through the association of their repeat regions. We hypothesize that the PthA variants might also interact with distinct host targets. Here, in addition to the interaction with alpha-importin, known to mediate the nuclear import of AvrBs3, we describe new interactions of PthAs with citrus proteins involved in protein folding and K63-linked ubiquitination. PthAs 2 and 3 preferentially interact with a citrus cyclophilin (Cyp) and with TDX, a tetratricopeptide domain-containing thioredoxin. In addition, PthAs 2 and 3, but not 1 and 4, interact with the ubiquitin-conjugating enzyme complex formed by Ubc13 and ubiquitin-conjugating enzyme variant (Uev), required for K63-linked ubiquitination and DNA repair. We show that Cyp, TDX and Uev interact with each other, and that Cyp and Uev localize to the nucleus of plant cells. Furthermore, the citrus Ubc13 and Uev proteins complement the DNA repair phenotype of the yeast Delta ubc13 and Delta mms2/uev1a mutants, strongly indicating that they are also involved in K63-linked ubiquitination and DNA repair. Notably, PthA 2 affects the growth of yeast cells in the presence of a DNA damage agent, suggesting that it inhibits K63-linked ubiquitination required for DNA repair.

Proteomic analysis reveals suppression of bark chitinases and proteinase inhibitors in citrus plants affected by the citrus sudden death disease

Citrus sudden death (CSD) is a disease of unknown etiology that greatly affects sweet oranges grafted on Rangpur lime rootstock, the most important rootstock in Brazilian citriculture. We performed a proteomic analysis to generate information related to this plant pathogen interaction. Protein profiles from healthy, CSD-affected and CSD-tolerant stem barks, were generated using two-dimensional gel electrophoresis. The protein spots were well distributed over a pI range of 3.26 to 9.97 and a molecular weight (MW) range from 7.1 to 120 kDa. The patterns of expressed proteins on 2-DE gels made it possible to distinguish healthy barks from CSD-affected barks. Protein spots with MW around 30 kDa and pI values ranging from 4.5 to 5.2 were down-regulated in the CSD-affected rootstock bark. This set of protein spots was identified as chitinases. Another set of proteins, ranging in pI from 6.1 to 9.6 with an MW of about 20 kDa, were also suppressed in CSD-affected rootstock bark; these were identified as miraculin-like proteins, potential trypsin inhibitors. Downregulation of chitinases and proteinase inhibitors in CSD-affected plants is relevant since chitinases are well-known pathogenesis-related protein, and their activity against plant pathogens is largely accepted.

Nuclear magnetic resonance characterization of metabolite disorder in orange trees caused by citrus sudden death disease

Citrus sudden death (CSD) is a new disease of sweet orange and mandarin trees grafted on Rangpur lime and Citrus volkameriana rootstocks. It was first seen in Brazil in 1999, and has since been detected in more than four million trees. The CSD causal agent is unknown and the current hypothesis involves a virus similar to Citrus tristeza virus or a new virus named Citrus sudden death-associated virus. CSD symptoms include generalized foliar discoloration, defoliation and root death, and, in most cases, it can cause tree death. One of the unique characteristics of CSD disease is the presence of a yellow stain in the rootstock bark near the bud union. This region also undergoes profound anatomical changes. In this study, we analyse the metabolic disorder caused by CSD in the bark of sweet orange grafted on Rangpur lime by nuclear magnetic resonance (NMR) spectroscopy and imaging. The imaging results show the presence of a large amount of non-functional phloem in the rootstock bark of affected plants. The spectroscopic analysis shows a high content of triacylglyceride and sucrose, which may be related to phloem blockage close to the bud union. We also propose that, without knowing the causal CSD agent, the determination of oil content in rootstock bark by low-resolution NMR can be used as a complementary method for CSD diagnosis, screening about 300 samples per hour.

Immobilized enzyme technology for debittering citrus fruit juices

There has been increased interest in the use of immobilized enzymes in fruit juice industry for debittering of citrus fruit juices due to their high efficiency to remove bitter flavonoids. The structure of naringin, responsible for immediate bitterness, and of limonin, responsible for "delayed bitterness" has been discussed. This chapter also discusses various attempts that have been made to immobilize enzymes on an appropriate support so as to enable their use in debittering of citrus fruit juices. These include physicochemical and enzyme biotechnological approaches which makes the fruit juice more acceptable and cost effective to the consumer. Despite of high volume of production of citrus fruits and fruit juices, suitable processes to produce non-bitter citrus juice by immobilized enzymes technology has not yet commercialized globally.

Sperm immobilizing properties of lemon juice

The aim of this study was to evaluate the sperm-immobilizing properties of lemon juice to determine if they are consistent with its traditional contraceptive use. It was found that lemon juice supernatant (LJS) has high osmolality (550–60 mOsm) and low pH (2.2–2.6) and that addition of LJS to semen to give a final concentration of 20% v/v reduced the pH from around 8.4 to 4.1. This acidification was associated with irreversible cessation of all sperm movements within 1 minute. In conclusion, lemon juice should be further evaluated for acceptability, safety, and efficacy as a topical vaginal contraceptive agent.

Citrus peel influences the production of an extracellular naringinase by Staphylococcus xylosus MAK2 in a stirred tank reactor

Staphylococcus xylosus MAK2, Gram-positive coccus, a nonpathogenic member of the coagulase-negative Staphylococcus family was isolated from soil and used to produce naringinase in a stirred tank reactor. An initial medium at pH 5.5 and a cultivation temperature of 30°C was found to be optimal for enzyme production. The addition of Ca⁺² caused stimulation of enzyme activity. The effect of various physico-chemical parameters, such as pH, temperature, agitation, and inducer concentration was studied. The enzyme production was enhanced by the addition of citrus peel powder (CPP) in the optimized medium. A twofold increase in naringinase production was achieved using different technological combinations. The process optimization using technological combinations allowed rapid optimization of large number of variables, which significantly improved enzyme production in a 5-l reactor in 34 h. An increase in sugar concentration (15 gl^-1) in the fermentation medium further increased naringinase production (8.9 IUml^-1) in the bioreactor. Thus, availability of naringinase renders it attractive for potential biotechnological applications in citrus processing industry.

Molecular characterization and enzymatic hydrolysis of flavanoid extracted from citrus waste

The citrus fruit processing industry generates substantial quantities of waste rich in phenolic substances, which is a valuable natural source of polyphenols (flavonoids) such as naringin and its disposal is becoming a major problem. In the US alone, the juice processing of oranges and grapefruit generates over 5 Mt of citrus waste every year. In the case of India, about 2.15 Mt of citrus peel out of 6.28 Mt of citrus fruits are produced yearly from citrus juice processing. In case of Australia, about 15-40% of citrus peel waste is generated by processing of citrus fruit (0.85 Mt). Thus Isolation of functional compounds (mostly flavanoids) and their further processing can be of interest to the food and pharmaceutical industry. This peel is rich in naringin and may be used for rhamnose production by utilizing α-L-rhamnosidase (EC 3.2.1.40), an enzyme that catalyzes the cleavage of terminal rhamnosyl groups from naringin to yield prunin and rhamnose. We recently purified recombinant α-L-rhamnosidase from E. coli cells using immobilized metal-chelate affinity chromatography (IMAC) and used it for naringin hydrolysis. The purified enzyme established hydrolysis of naringin extracted from citrus peel and thus endorses its industrial applicability for producing rhamnose. Infrared (IR) spectroscopy confirmed molecular characteristics of naringin extracted from citrus peel waste.

Hydrolysis of citrus peel waste with recombinant rhamnosidase for bioenergy generation

Large amounts of Citrus peel (rich in poly-phenolic compounds) are generated as a by-product of the juice processing industry. Development of alternative, higher valued products utilizing peel waste from grapefruit, oranges, Valencia and other citrus fruit would benefit citrus juice processors by providing them with means to profitably process their peel waste and to avoid environmentally hazardous dumping. Citrus peel waste [CPW, comprised of peel, membranes and juice vesicles] contains a high level of polyphenols and has been used for the production of animal feed, single-cell protein, fibre, enzyme(s), immobilization support & bio-sorbent for heavy metal removal. Naringin (a major tri-hydroxy flavonoid glycoside) is available in large amounts in citrus peel, processed juice and can be extracted from citrus peel waste1. The extracted naringin is further hydrolysed by rhamnosidase to produce D-rhamnose for the production of ethanol and other fermentation products. We have produced a recombinant enzyme2 that has the ability to catalyse the cleavage of terminal rhamnoside groups from naringin to prunin and rhamnose. We have recovered important sugar “D-rhamnose” from the processed waste which would be utilized for ethanol production3. This presentation will summarize current efforts to develop an enzymatic treatment which would facilitate the economical processing of citrus waste for bioenergy generation.

O papel das tecnologias de informação e comunicação na oferta de serviços financeiros para a população de baixa renda : o caso dos correspondentes bancários do Banco Lemon

Algumas abordagens recentes chamaram a atenção para a importância das empresas mobilizarem sua capacidade produtiva para solucionar problemas das classes menos favorecidas da sociedade. Especificamente para o caso de serviços financeiros, defende-se que a atuação na baixa renda tem implicações positivas na sociedade como um todo, ao promover sua re-inclusão no sistema tradicional. Dentro desse cenário, as iniciativas de microfinanças parecem jogar luz a esse problema ao tentar levar acesso a serviços financeiros para a população de baixa renda. Porém, tais iniciativas ainda parecem não ter demonstrado o seu real potencial, e prova disso é a persistência dos crônicos indicadores de pobreza e de desigualdade social. Isso acontece porque as abordagens atuais de microfinanças apresentam limitações, dentre as quais as tecnológicas são uma das mais importantes. Por outro lado, existe um fenômeno que se manifesta especificamente no Brasil e que se refere ao grande crescimento dos chamados correspondentes bancários (CBs), em que o correspondente, que, na maior parte das vezes, é um varejo, oferta serviços em nome do banco. Alguns estudos relacionam esses dois conceitos, microfinanças e CBs, e propõem o CB como um canal tecnológico para a promoção das microfinanças. Dentro desse cenário, uma pergunta pertinente e ainda pouco explorada na literatura é: como ocorrem a adoção, implementação e uso das tecnologias de correspondentes bancários na oferta de serviços financeiros para a população de baixa renda no Brasil? De posse dessa pergunta de pesquisa, procedeu-se a um estudo de caso em profundidade e o objeto de estudo foi a forma de atuação do Banco Lemon, em especial, por possuir uma extensa rede de CBs, ser baseado exclusivamente em CBs (não tem agências físicas) e estar focado na baixa renda. A partir de uma abordagem que inclui contribuições das escolas do estruturacionismo, do construtuvismo e do contextualismo, foi possível entender como ocorreu o processo de negociação entre dois grupos relevantes, o Banco Lemon e o gestor de redes, que é um intermediário entre o banco e o varejo, na formação da tecnologia que foi adotada, implementada e usada. Os principais achados da pesquisa referem-se ao entendimento de como a tecnologia é usada para prover os serviços que o Banco Lemon quer ofertar e como dois grupos relevantes interagem, negociam e produzem a tecnologia na prática.