Novel broad-spectrum bacteriophages against Xanthomonas oryzae and their biocontrol potential in rice bacterial diseases

Bacterial leaf blight (BLB) and bacterial leaf streak (BLS)—caused by Xanthomonas oryzae pv. oryzae (Xoo) and Xanthomonas oryzae pv. oryzicola (Xoc), respectively—are two major bacterial diseases that threaten the safe production of rice, one of the most important food crops. Bacteriophages are considered potential biocontrol agents against rice bacterial pathogens, due to their host specificity and environmental safety. It is common for BLB and BLS to occur together in fields, which highlights the need for broad-spectrum phages capable of infecting both Xoo and Xoc. In this study, two lytic broad-spectrum phages (pXoo2106 and pXoo2107) that can infect various strains of Xoo and Xoc were assessed. Both phages belong to the class Caudoviricetes and one of them to the family Autographiviridae, while the other belongs to an unclassified family. Two phages alone or combined in a phage cocktail could effectively inhibit Xoo and Xoc growth in vitro. In an in vivo biocontrol experiment, the phage cocktail reduced the total CFU and significantly eased the symptoms caused by Xoo or Xoc. Our results suggest that pXoo2106 and pXoo2107 have a broad-spectrum host range targeting different X. oryzae strains, and have strong biocontrol potential in field applications against both BLB and BLS.     

Highly efficient genome editing in Xanthomonas oryzae pv. oryzae through repurposing the endogenous type I-C CRISPR-Cas system

Efficient and modular genome editing technologies that manipulate the genome of bacterial pathogens will facilitate the study of pathogenesis mechanisms. However, such methods are yet to be established for Xanthomonas oryzae pv. oryzae (Xoo), the causal agent of rice bacterial blight. We identified a single type I-C CRISPR-Cas system in the Xoo genome and leveraged this endogenous defence system for high-efficiency genome editing in Xoo. Specifically, we developed plasmid components carrying a mini-CRISPR array, donor DNA, and a phage-derived recombination system to enable the efficient and programmable genome editing of precise deletions, insertions, base substitutions, and gene replacements. Furthermore, the type I-C CRISPR-Cas system of Xoo cleaves target DNA unidirectionally, and this can be harnessed to generate large genomic deletions up to 212 kb efficiently. Therefore, the genome-editing strategy we have developed can serve as an excellent tool for functional genomics of Xoo, and should also be applicable to other CRISPR-harbouring bacterial plant pathogens.     

RIR1 represses plant immunity by interacting with mitochondrial complex I subunit in rice

We previously observed decreased expression of rice OsmiR159a.1 on infection with the bacterial blight-causing pathogen Xanthomonas oryzae pv. oryzae (Xoo), and identified the OsLRR_RLK (leucine-rich repeat_ receptor like kinase) gene as an authentic target of OsmiR159a.1. Here, we found that a Tos17 insertion mutant of LRR_RLK displayed increasing temporal resistance to Xoo, whereas the LRR_RLK overexpression lines were susceptible to the pathogen early on in the infection, indicating that LRR_RLK encodes a repressor of rice resistance to Xoo infection, and it was renamed as RIR1 (Rice Immunity Repressor 1). RIR1 overexpression plants were more susceptible to Xoo at late growth stage, suggesting that RIR1 mRNA levels are negatively correlated with the resistance of rice against Xoo. We discovered that OsmiR159a.1 repression in Xoo-infected plants was largely dependent on the pathogen's type III secretion system. Co-immunoprecipitation, bimolecular fluoresence complementation, and pull-down assays indicated that RIR1 interacted with the NADH-ubiquinone oxidoreductase (NUO) 51-kDa subunit of the mitochondrial complex I through its kinase domain. Notably, impairment of RIR1 or overexpression of NUO resulted in reactive oxygen species accumulation and enhanced expression of pathogen-resistance genes, including jasmonic acid pathway genes. We propose that pathogens may inhibit OsmiR159 to interfere with the RIR1–NUO interaction, and subsequently depression of rice immune signalling pathways. The resistance genes manipulated by Xoo can be a probe to explore the regulatory network during host–pathogen interactions.     

Constitutive heterologous expression of avrXa27 in rice containing the R gene Xa27 confers enhanced resistance to compatible Xanthomonas oryzae strains

The vascular pathogen Xanthomonas oryzae pv. oryzae ( Xoo ) and nonvascular pathogen Xanthomonas oryzae pv. oryzicola ( Xoc ) cause bacterial blight (BB) and bacterial leaf streak (BLS) diseases of rice, respectively. We have previously identified the avirulence gene avrXa27 from Xoo PXO99^A, which specifically induces the expression of the rice resistance gene Xa27 , ultimately leading to resistance against BB disease in rice. In this study, we have generated a transgenic rice line (L24) that expresses avrXa27 constitutively under the control of the PR1 promoter, and have examined its role in the host–pathogen interaction. L24 is not more susceptible to BB, indicating that avrXa27 does not contribute to virulence. AvrXa27 retains avirulence activity in L24 and, after crossing with a line containing Xa27 , progeny display phenotypic changes including inhibition of tillering, delay in flowering, stiff leaves, early leaf senescence and activation of pathogenesis-related ( PR ) genes. On challenge with a variety of compatible strains of Xoo and Xoc strain L8, lines with both avrXa27 and Xa27 also show enhanced resistance to bacterial infection. The induction of Xa27 and subsequent inhibition of Xoc growth in Xa27 plants are observed on inoculation with Xoc L8 harbouring avrXa27 . Our results indicate that the heterologous expression of avrXa27 in rice containing Xa27 triggers R gene-specific resistance and, at the same time, confers enhanced resistance to compatible strains of Xoo and Xoc . The expression of AvrXa27 and related proteins in plants has the potential to generate broad resistance in plants.     

A set of modular plant transformation vectors allowing flexible insertion of up to six expression units

We have constructed a binary vector for Agrobacterium-mediated plant transformation, which has a multiple cloning site consisting of 13 hexanucleotide restriction sites, 6 octanucleotide restriction sites and 5 homing endonuclease sites. The homing endonuclease sites have the advantages to be extremely rare in natural sequences and to allow unidirectional cloning. We have also constructed a set of auxiliary vectors allowing the assembly of expression cassettes flanked by homing endonuclease sites. The expression cassettes assembled in these auxiliary vectors can be transferred into the binary vector with virtually no risk of cutting the vector within previously introduced sequences. This vector set is ideally suited for the construction of plant transformation vectors containing multiple expression cassettes and/or other elements such as matrix attachment regions. With this modular vector system, six different expression units were constructed in as many auxiliary vectors and assembled together in one plant transformation vector. The transgenic nature of Arabidopsis thaliana plants, transformed with this plant transformation vector, was assessed and the expression of each of the six genes was demonstrated.     

Domain Dissection of AvrRxo1 for Suppressor,Avirulence and Cytotoxicity Functions

AvrRxo1, a type III effector from Xanthomonas oryzae pv. oryzicola (Xoc) which causes bacterial leaf streak (BLS) in rice, can be recognised by non-host resistance protein Rxo1. It triggers a hypersensitive response (HR) in maize. Little is known regarding the virulence function of AvrRxo1. In this study, we determined that AvrRxo1 is able to suppress the HR caused by the non-host resistance recognition of Xanthomonas oryzae pv. oryzae (Xoo) by Nicotiana benthamiana. It is toxic, inducing cell death from transient expression in N. benthamiana, as well as in yeast. Among the four AvrRxo1 alleles from different Xoc strains, we concluded that the toxicity is abolished by a single amino acid substitution at residue 344 in two AvrRxo1 alleles. A series of truncations from the carboxyl terminus (C-terminus) indicate that the complete C-terminus of AvrRxo1 plays an essential role as a suppressor or cytotoxic protein. The C-terminus was also required for the avirulence function, but the last two residues were not necessary. The first 52 amino acids of N-terminus are unessential for toxicity. Point mutagenesis experiments indicate that the ATP/GTP binding site motif A is required for all three functions of AvrRxo1, and NLS is required for both the avirulence and the suppression of non-host resistance. The putative thiol protease site is only required for the cytotoxicity function. These results determine that AvrRxo1 plays a role in the complex interaction with host proteins after delivery into plant cells.     

Multilocus sequence analysis and type III effector repertoire mining provide new insights into the evolutionary history and virulence of Xanthomonas oryzae

Multilocus sequence analysis (MLSA) and type III effector (T3E) repertoire mining were performed to gain new insights into the genetic relatedness of Xanthomonas oryzae pv. oryzae (Xoo) and Xanthomonas oryzae pv. oryzicola (Xoc), two major bacterial pathogens of rice. Based on a collection of 45 African and Asian strains, we first sequenced and analysed three housekeeping genes by MLSA, Bayesian clustering and a median-joining network approach. Second, we investigated the distribution of 32 T3E genes, which are known to be major virulence factors of plant pathogenic bacteria, in all selected strains, by polymerase chain reaction and dot-blot hybridization methods. The diversity observed within housekeeping genes, as well as within T3E repertoires, clearly showed that both pathogens belong to closely related, but distinct, phylogenetic groups. Interestingly, these evolutionary groups are differentiated according to the geographical origin of the strains, suggesting that populations of Xoo and Xoc might be endemic in Africa and Asia, and thus have evolved separately. We further revealed that T3E gene repertoires of both pathogens comprise core and variable gene suites that probably have distinct roles in pathogenicity and different evolutionary histories. In this study, we carried out a functional analysis of xopO, a differential T3E gene between Xoo and Xoc, to determine the involvement of this gene in tissue specificity. Altogether, our data contribute to a better understanding of the evolutionary history of Xoo and Xoc in Africa and Asia, and provide clues for functional studies aiming to understand the virulence, host and tissue specificity of both rice pathogens.     

Transient expression of heterologous model gene in plants using <Emphasis Type="Italic">Potato virus</Emphasis><Emphasis Type="Italic">X</Emphasis>-based vector

To optimize the efficiency of expression of foreign proteins using Potato virus X (PVX) -- based vector, the gene for the coat protein (CP) of other virus (Potato virus A, PVA) was cloned into the vector, propagated in E. coli and subsequently inoculated or agroinfected into the host plants. Host range studies showed that the best host plant is N. benthamiana. By means of RT PCR the presence and the stability of the construct were tested. Both ELISA and Western blot analysis were applicable for expressed protein detection. Expression level of PVA CP achieved approximately 5--10 per mille of total soluble proteins. The results demonstrated that agroinfection is the most suitable method for the propagation of our model gene using PVX--based vectors.     

Cloning Vectors for Rice

We developed various binary vectors that can be used for expressing a foreign gene in rice. Vectors pGA3426, pGA3436, and pGA3626 are intended for overexpression of a gene using the maize Ubiquitin promoter, whereas pGA3780 is for rather mild expression of a gene using the rice Actin1 promoter. Vector pGA3777 is for expressing two genes simultaneously. We also developed binary vectors for expressing a fusion protein with a tag. Four vectors (pGA3427, pGA3428, pGA3429, and pGA3438) are for protein tags with sGFP, HA, His, and Myc, respectively. Vector pGA3383 is for analyzing promoter activity using the GUS reporter. In this vector, multiple cloning sites in front of GUS can be utilized for accepting a promoter fragment. We also generated transient expression vectors for studying the subcellular localization of a protein. Vectors pGA3452, pGA3651, and pGA3652 are for GFP fusion; pGA3574 for RFP fusion; pGA3697 for Myc tag; and pGA3698 for HA tag. In addition, we generated pGA3506, pGA3516, pGA3592, and pGA3593 for facilitating the subcloning of full-length cDNA clones into our binary vectors.     

Role of the FnIII domain associated with a cell wall-degrading enzyme cellobiosidase of Xanthomonas oryzae pv. oryzae

Cellobiosidase (CbsA) is an important secreted virulence factor of Xanthomonas oryzae pv. oryzae (Xoo), which causes bacterial blight of rice. CbsA is one of several cell wall-degrading enzymes secreted by Xoo via the type II secretion system (T2SS). CbsA is considered a fundamental virulence factor for vascular pathogenesis. CbsA has an N-terminal glycosyl hydrolase domain and a C-terminal fibronectin type III (FnIII) domain. Interestingly, the secreted form of CbsA lacks the FnIII domain during in planta growth. Here we show that the presence of the FnIII domain inhibits the enzyme activity of CbsA on polysaccharide substrates like carboxymethylcellulose. The FnIII domain is required for the interaction of CbsA with SecB chaperone, and this interaction is crucial for the stability and efficient transport of CbsA across the inner membrane. Deletion of the FnIII domain reduced virulence similar to ΔcbsA Xoo, which corroborates the importance of the FnIII domain in CbsA. Our work elucidates a hitherto unknown function of the FnIII domain in enabling the virulence-promoting activity of CbsA.     

A Versatile System for USER Cloning-Based Assembly of Expression Vectors for Mammalian Cell Engineering

A new versatile mammalian vector system for protein production, cell biology analyses, and cell factory engineering was developed. The vector system applies the ligation-free uracil-excision based technique – USER cloning – to rapidly construct mammalian expression vectors of multiple DNA fragments and with maximum flexibility, both for choice of vector backbone and cargo. The vector system includes a set of basic vectors and a toolbox containing a multitude of DNA building blocks including promoters, terminators, selectable marker- and reporter genes, and sequences encoding an internal ribosome entry site, cellular localization signals and epitope- and purification tags. Building blocks in the toolbox can be easily combined as they contain defined and tested Flexible Assembly Sequence Tags, FASTs. USER cloning with FASTs allows rapid swaps of gene, promoter or selection marker in existing plasmids and simple construction of vectors encoding proteins, which are fused to fluorescence-, purification-, localization-, or epitope tags. The mammalian expression vector assembly platform currently allows for the assembly of up to seven fragments in a single cloning step with correct directionality and with a cloning efficiency above 90%. The functionality of basic vectors for FAST assembly was tested and validated by transient expression of fluorescent model proteins in CHO, U-2-OS and HEK293 cell lines. In this test, we included many of the most common vector elements for heterologous gene expression in mammalian cells, in addition the system is fully extendable by other users. The vector system is designed to facilitate high-throughput genome-scale studies of mammalian cells, such as the newly sequenced CHO cell lines, through the ability to rapidly generate high-fidelity assembly of customizable gene expression vectors.