Table_1_A root-knot nematode effector manipulates the rhizosphere microbiome for establishing parasitism relationship with hosts.DOCX

IntroductionRoot-knot nematode (RKN; Meloidogyne spp.) is one of the most infamous soilborne plant diseases, causing severe crop losses every year. Effector proteins secreted by RKNs play crucial roles during plant-nematode interaction. However, less is known about whether RKN effector proteins can impact the rhizosphere microbial environment.MethodsIn this study, we investigated the rhizosphere microbiome community of MiMIF-2 (a plant immunity-modulating effector) transgenic Arabidopsis thaliana with or without nematode infection using the Illumina high-throughput sequencing analysis.Results and discussionThe results showed that the bacterial species richness index increased, while the fungi species richness index decreased in M. incognita-infected MiMIF-2 transgenic A. thaliana plants. The relative abundance of genera such as Clitopilus, Komagataeibacter, Lactobacillus, Prevotella, Moritella, Vibrio, Escherichia-Shigella, and Pseudomonas was reduced in MiMIF-2 transgenic A. thaliana plants compared to wild type, but was significantly increased after inoculation with M. incognita. The Cluster of Orthologous Genes (COG) function classification analysis revealed a decrease in the relative abundance of defense mechanisms, secondary metabolite biosynthesis, transport, and nematode infection catabolism-related functions in MiMIF-2 lines compared to the wild type. These differences may be the reason for the increased susceptibility of MiMIF-2 transgenic A. thaliana to nematode infection. Our results provide a new insight into RKN effector proteins and their association with the microbial community, host, and plant pathogens, which will lead to the exploration of new innovative ideas for future biological control of RKNs.</p

Highly Sensitive and Selective Chip-Based Fluorescent Sensor for Mercuric Ion: Development and Comparison of Turn-On and Turn-Off Systems

Miniaturization is currently an important trend in environmental and food monitoring because it holds great promise for on-site monitoring and detection. We report here two ready-to-use chip-based fluorescent sensors, compatible with microarray technology for reagentless, one-step, fast, highly sensitive and selective detection of the mercuric ion (Hg²⁺) in the turn-on and turn-off operation modes. Both operation modes are based on the highly selective T–Hg²⁺–T coordination between two neighboring polythymine (T) strands at a high probe density and its induced displacement of the complementary polyadenine strand labeled with either fluorophore or quencher, which enables the turn-off and turn-on detection of Hg²⁺, respectively. The turn-off sensor is slightly more sensitive than the turn-on sensor, and their detection limits are 3.6 and 8.6 nM, respectively, which are both lower than the U.S. Environmental Protection Agency limit of [Hg²⁺] for drinkable water (10 nM, 2 ppb). Compared to the turn-off sensor with the dynamic Hg²⁺ detection range from 3.6 nM to 10 μM (<i>R</i>² = 0.99), the turn-on sensor has a broader dynamic Hg²⁺ detection range, from 8.6 nM to 100 μM (<i>R</i>² = 0.996). Both sensors exhibited superior selectivity over other reported sensors using thymine-rich probes for Hg²⁺ detection over other common metal ions. In addition, the practical application of the chip-based sensors was demonstrated by detecting spiked Hg²⁺ in drinking water and fresh milk. The sensor has great potential for on-site practical applications due to its operational convenience, simplicity, speed, and portability

Suppression of BT-PCD by Ha-ANNEXIN.

<p>(A) Assay for the suppression of BAX-triggered cell death (BT-PCD) in <i>Nicotiana benthamiana</i> by Ha-ANNEXIN. <i>N</i>. <i>benthamiana</i> leaves were infiltrated with buffer or <i>Agrobacterium tumefaciens</i> cells containing a vector carrying the <i>Ha-annexin</i> gene or the negative control <i>eGFP</i> gene, either alone or followed 24 h later with <i>A</i>. <i>tumefaciens</i> cells carrying a mouse <i>Bax</i> gene. Photos of phenotypes of infiltrated leaves of <i>N</i>. <i>benthamiana</i> were taken approximately 7 days after the last infiltration. Results of the verification of gene expression of <i>Ha-annexin</i>, <i>eGFP</i> and <i>Bax</i> by RT-PCR or western blotting are shown below. (B) Necrosis Index of Ha-ANNEXIN and control eGFP followed by BAX. Each column represents the mean with standard deviation. The column with asterisks indicate a highly statistically significant reduction of the Necrosis Index of Ha-ANNEXIN compared with that of eGFP by t-test (<i>P</i><0.01).</p

Ha-ANNEXIN suppresses flg22-triggered upregulation of PTI marker genes in <i>Nicotiana benthamiana</i>.

<p>The upregulation of three PTI marker genes—<i>NbPti5</i>, <i>NbAcre31</i> and <i>NbGras2</i>—after flg22 treatment in <i>N</i>. <i>benthamiana</i> leaf tissues expressing Ha-ANNEXIN or the negative control eGFP was compared using qPCR, respectively. Each column represents the mean with standard deviation. The column with asterisks indicate a highly statistically significant difference compared with the eGFP negative control by t-test (<i>P</i><0.01).</p

Multiple alignment of annexins from <i>Heterodera avenae</i> and some other plant-parasitic nematodes.

<p>In the consensus, residues in the black background are totally identical; boxed areas are four conserved annexin domains predicted by the NCBI Conserved Domain Search Service; signal peptides of <i>H</i>. <i>schachtii</i> and <i>H</i>. <i>glycines</i> analyzed by Signal P 4.0 are underlined. SP, signal peptide; Gp, <i>Globodera pallida</i>; Bx, <i>Bursaphelenchus xylophilus</i>; Hs, <i>H</i>. <i>schachtii</i>; Hg, <i>H</i>. <i>glycines</i>.</p

Subcellular localization of Ha-ANNEXIN in the plant cell.

<p>(A) pUC35S:ANNEXIN:GFP fusion construct and pUC35S:GFP control construct were transformed into onion epidermal cells by bombardment. Scale bar = 100 μm. (B) <i>Agrobacterium tumefaciens</i> cells carrying pCamv35S:ANNEXIN:GFP fusion and pCamv35S:GFP were transiently expressed in <i>Nicotiana benthamiana</i> cells. Scale bar = 20 μm. Western blotting of <i>N</i>. <i>benthamiana</i> leaves infiltrated with pCamv35SGFP-annexin showed expected size of annexin-GFP fusion, which is larger than GFP control. In both (A) and (B), GFP signals were observed in the whole transformed cells for annexin-GFP fusion, which is the same as GFP control.</p

Effect of BSMV-HIGS of <i>Ha-annexin</i> on infection of wheat roots by <i>H</i>. <i>avenae</i>.

<p>(A) At 7 dpi, the expression of <i>Ha-annexin</i> in nematodes recovered from wheat inoculated by BSMV:<i>annexin</i> was not detected by qPCR with BSMV:00 and BSMV:<i>eGFP</i> as positive controls. Nematode infection of wheat inoculated by BSMV:<i>annexin</i> compared to the blank negative control BSMV:00 and the negative control BSMV:<i>eGFP</i> showed a highly significant reduction in the number of juveniles/plant at 7 dpi (B) and females/plant at 40 dpi (C) by Duncan test (<i>P</i><0.01). Each column represents the mean with standard deviation.</p

Ha-ANNEXIN can suppress MAPK-triggered cell death.

<p>Ha-ANNEXIN can suppress MKK1 (A)—or NPK1^Nt (B)-triggered cell death. Results of the verification of expression of <i>annexin and eGFP</i> by western blotting are shown below. The Necrosis Index of Ha-ANNEXIN and control eGFP followed by MKK1 (A) or NPK1^Nt (B) was scored. Each column represents the mean with standard deviation. The column with asterisks indicate a highly statistically significant reduction of the Necrosis Index of Ha-ANNEXIN compared with that of eGFP by t-test (<i>P</i><0.01).</p

Southern blot, <i>in situ</i> hybridization and developmental expression pattern analysis of <i>Ha-annexin</i>.

<p>(A) Southern blot analysis of <i>Ha-annexin</i>. Genomic DNA from <i>H</i>. <i>avenae</i> was digested with <i>Bam</i>HI and <i>Eco</i>RI, respectively, and probed with the Dig-labeled CDS of <i>Ha-annexin</i>. They had 3 and 2 signal strips, respectively. (B) <i>In situ</i> hybridization of the <i>Ha-annexin</i> transcripts in pre-parasitic second-stage juveniles. Signal of antisense <i>Ha-annexin</i> DIG-Labeled cDNA probes localized within the subventral glands (SVGs), with sense probes as a negative control. The SVG, metacorpus (M), and stylet (S) are indicated with arrows. Scale bar = 20 μm. (C) Developmental expression pattern of <i>Ha-annexin</i>. The relative expression level of <i>Ha-annexin</i> was quantified using qPCR for six different <i>H</i>. <i>avenae</i> stages. The fold change values were calculated using the 2^-ΔΔCt method and presented as the change in mRNA level in various nematode developmental stages relative to that of egg. Each column represents the mean of 3 independent assays with standard deviation. preJ2: pre-parasitic second-stage juvenile; parJ2, J3 and J4: parasitic second-, third- and fourth-stage juvenile, respectively.</p

An ANNEXIN-Like Protein from the Cereal Cyst Nematode <i>Heterodera avenae</i> Suppresses Plant Defense

<div><p>Parasitism genes encoding secreted effector proteins of plant-parasitic nematodes play important roles in facilitating parasitism. An annexin-like gene was isolated from the cereal cyst nematode <i>Heterodera avenae</i> (termed <i>Ha-annexin</i>) and had high similarity to <i>annexin 2</i>, which encodes a secreted protein of <i>Globodera pallida</i>. <i>Ha-annexin</i> encodes a predicted 326 amino acid protein containing four conserved annexin domains. Southern blotting revealed that there are at least two homologies in the <i>H</i>. <i>avenae</i> genome. <i>Ha-annexin</i> transcripts were expressed within the subventral gland cells of the pre-parasitic second-stage juveniles by <i>in situ</i> hybridization. Additionally, expression of these transcripts were relatively higher in the parasitic second-stage juveniles by quantitative real-time RT-PCR analysis, coinciding with the time when feeding cell formation is initiated. Knockdown of <i>Ha-annexin</i> by method of barley stripe mosaic virus-based host-induced gene silencing (BSMV-HIGS) caused impaired nematode infections at 7 dpi and reduced females at 40 dpi, indicating important roles of the gene in parasitism at least in early stage <i>in vivo</i>. Transiently expression of Ha-ANNEXIN in onion epidermal cells and <i>Nicotiana benthamiana</i> leaf cells showed the whole cell-localization. Using transient expression assays in <i>N</i>. <i>benthamiana</i>, we found that Ha-ANNEXIN could suppress programmed cell death triggered by the pro-apoptotic mouse protein BAX and the induction of marker genes of PAMP-triggered immunity (PTI) in <i>N</i>. <i>benthamiana</i>. In addition, Ha-ANNEXIN targeted a point in the mitogen-activated protein kinase (MAPK) signaling pathway downstream of two kinases MKK1 and NPK1 in <i>N</i>. <i>benthamiana</i>.</p></div